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Sample records for nonribosomal peptide synthases

  1. Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

    Science.gov (United States)

    Luo, Chuping; Liu, Xuehui; Zhou, Huafei; Wang, Xiaoyu; Chen, Zhiyi

    2015-01-01

    Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

  2. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

    Directory of Open Access Journals (Sweden)

    Grigoris D. Amoutzias

    2016-04-01

    Full Text Available Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs and polyketides (PKs are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes and type-I polyketide synthases (PKSes-I, respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

  3. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes.

    Science.gov (United States)

    Amoutzias, Grigoris D; Chaliotis, Anargyros; Mossialos, Dimitris

    2016-04-16

    Considering that 70% of our planet's surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

  4. Insect-specific polyketide synthases (PKSs), potential PKS-nonribosomal peptide synthetase hybrids, and novel PKS clades in tropical fungi.

    Science.gov (United States)

    Amnuaykanjanasin, Alongkorn; Phonghanpot, Suranat; Sengpanich, Nattapong; Cheevadhanarak, Supapon; Tanticharoen, Morakot

    2009-06-01

    Polyketides draw much attention because of their potential use in pharmaceutical and biotechnological applications. This study identifies an abundant pool of polyketide synthase (PKS) genes from local isolates of tropical fungi found in Thailand in three different ecological niches: insect pathogens, marine inhabitants, and lichen mutualists. We detected 149 PKS genes from 48 fungi using PCR with PKS-specific degenerate primers. We identified and classified 283 additional PKS genes from 13 fungal genomes. Phylogenetic analysis of all these PKS sequences the comprising ketosynthase (KS) conserved region and the KS-acyltransferase interdomain region yielded results very similar to those for phylogenies of the KS domain and suggested a number of remarkable points. (i) Twelve PKS genes amplified from 12 different insect-pathogenic fungi form a tight cluster, although along with two PKS genes extracted from genomes of Aspergillus niger and Aspergillus terreus, in reducing clade III. Some of these insect-specific fungal PKSs are nearly identical. (ii) We identified 38 new PKS-nonribosomal peptide synthetase hybrid genes in reducing clade II. (iii) Four distinct clades were discovered with more than 75% bootstrap support. We propose to designate the novel clade D1 with 100% bootstrap support "reducing clade V." The newly cloned PKS genes from these tropical fungi should provide useful and diverse genetic resources for future research on the characterization of polyketide compounds synthesized by these enzymes.

  5. Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains.

    Science.gov (United States)

    Keating, T A; Marshall, C G; Walsh, C T

    2000-12-19

    The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain. VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.

  6. Type I pyridoxal 5'-phosphate dependent enzymatic domains embedded within multimodular nonribosomal peptide synthetase and polyketide synthase assembly lines.

    Science.gov (United States)

    Milano, Teresa; Paiardini, Alessandro; Grgurina, Ingeborg; Pascarella, Stefano

    2013-10-23

    Pyridoxal 5'-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes. An ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context. This work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand

  7. Dissecting and Exploiting Nonribosomal Peptide Synthetases

    Institute of Scientific and Technical Information of China (English)

    Qing-Tao SHEN; Xiu-Lan CHEN; Cai-Yun SUN; Yu-Zhong ZHANG

    2004-01-01

    A large number of therapeutically useful cyclic and linear peptides of bacteria or fungal origin are synthesized via a template-directed, nucleic-acid-independent nonribosomal mechanism. This process is carried out by mega-enzymes called nonribosomal peptide synthetases (NRPSs). NRPSs contain repeated coordinated groups of active sites called modules, and each module is composed of several domains with different catalytic activities. The familiarity to these domains lays base for the future genetic engineering of NRPSs to generate entirely "unnature" Products. The details about NRPSs domain structures and the exploitation of NRPSs are described in this review.

  8. Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi.

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    Soukup, Alexandra A; Keller, Nancy P; Wiemann, Philipp

    2016-01-01

    Filamentous fungi are historically known as rich sources for production of biologically active natural products, so-called secondary metabolites. One particularly pharmaceutically relevant chemical group of secondary metabolites is the nonribosomal peptides synthesized by nonribosomal peptide synthetases (NRPSs). As most of the fungal NRPS gene clusters leading to production of the desired molecules are not expressed under laboratory conditions, efforts to overcome this impediment are crucial to unlock the full chemical potential of each fungal species. One way to activate these silent clusters is by overexpressing and deleting global regulators of secondary metabolism. The conserved fungal-specific regulator of secondary metabolism, LaeA, was shown to be a valuable target for sleuthing of novel gene clusters and metabolites. Additionally, modulation of chromatin structures by either chemical or genetic manipulation has been shown to activate cryptic metabolites. Furthermore, NRPS-derived molecules seem to be affected by cross talk between the specific gene clusters and some of these metabolites have a tissue- or developmental-specific regulation. This chapter summarizes how this knowledge of different tiers of regulation can be combined to increase production of NRPS-derived metabolites in fungal species.

  9. Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi

    Science.gov (United States)

    Soukup, Alexandra A.; Keller, Nancy P.; Wiemann, Philipp

    2016-01-01

    Filamentous fungi are historically known as rich sources for production of biologically active natural products, so-called secondary metabolites. One particularly pharmaceutically relevant chemical group of secondary metabolites is the nonribosomal peptides synthesized by nonribosomal peptide synthetases (NRPSs). As most of the fungal NRPS gene clusters leading to production of the desired molecules are not expressed under laboratory conditions, efforts to overcome this impediment are crucial to unlock the full chemical potential of each fungal species. One way to activate these silent clusters is by overexpressing and deleting global regulators of secondary metabolism. The conserved fungal-specific regulator of secondary metabolism, LaeA, was shown to be a valuable target for sleuthing of novel gene clusters and metabolites. Additionally, modulation of chromatin structures by either chemical or genetic manipulation has been shown to activate cryptic metabolites. Furthermore, NRPS-derived molecules seem to be affected by cross talk between the specific gene clusters and some of these metabolites have a tissue- or developmental-specific regulation. This chapter summarizes how this knowledge of different tiers of regulation can be combined to increase production of NRPS-derived metabolites in fungal species. PMID:26831707

  10. Structural pattern matching of nonribosomal peptides

    Directory of Open Access Journals (Sweden)

    Leclère Valérie

    2009-03-01

    Full Text Available Abstract Background Nonribosomal peptides (NRPs, bioactive secondary metabolites produced by many microorganisms, show a broad range of important biological activities (e.g. antibiotics, immunosuppressants, antitumor agents. NRPs are mainly composed of amino acids but their primary structure is not always linear and can contain cycles or branchings. Furthermore, there are several hundred different monomers that can be incorporated into NRPs. The NORINE database, the first resource entirely dedicated to NRPs, currently stores more than 700 NRPs annotated with their monomeric peptide structure encoded by undirected labeled graphs. This opens a way to a systematic analysis of structural patterns occurring in NRPs. Such studies can investigate the functional role of some monomeric chains, or analyse NRPs that have been computationally predicted from the synthetase protein sequence. A basic operation in such analyses is the search for a given structural pattern in the database. Results We developed an efficient method that allows for a quick search for a structural pattern in the NORINE database. The method identifies all peptides containing a pattern substructure of a given size. This amounts to solving a variant of the maximum common subgraph problem on pattern and peptide graphs, which is done by computing cliques in an appropriate compatibility graph. Conclusion The method has been incorporated into the NORINE database, available at http://bioinfo.lifl.fr/norine. Less than one second is needed to search for a pattern in the entire database.

  11. Recent advances in engineering nonribosomal peptide assembly lines.

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    Winn, M; Fyans, J K; Zhuo, Y; Micklefield, J

    2016-02-01

    Nonribosomal peptides are amongst the most widespread and structurally diverse secondary metabolites in nature with many possessing bioactivity that can be exploited for therapeutic applications. Due to the major challenges associated with total- and semi-synthesis, bioengineering approaches have been developed to increase yields and generate modified peptides with improved physicochemical properties or altered bioactivity. Here we review the major advances that have been made over the last decade in engineering the biosynthesis of nonribosomal peptides. Structural diversity has been introduced by the modification of enzymes required for the supply of precursors or by heterologous expression of tailoring enzymes. The modularity of nonribosomal peptide synthetase (NRPS) assembly lines further supports module or domain swapping methodologies to achieve changes in the amino acid sequence of nonribosomal peptides. We also review the new synthetic biology technologies promising to speed up the process, enabling the creation and optimisation of many more assembly lines for heterologous expression, offering new opportunities for engineering the biosynthesis of novel nonribosomal peptides.

  12. Characterization and localization of a hybrid non-ribosomal peptide synthetase and polyketide synthase gene from the toxic dinoflagellate Karenia brevis.

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    López-Legentil, Susanna; Song, Bongkeun; DeTure, Michael; Baden, Daniel G

    2010-02-01

    The toxic dinoflagellate Karenia brevis, a causative agent of the red tides in Florida, produces a series of toxic compounds known as brevetoxins and their derivatives. Recently, several putative genes encoding polyketide synthase (PKS) were identified from K. brevis in an effort to elucidate the genetic systems involved in brevetoxin production. In this study, novel PKS sequences were isolated from three clones of K. brevis. Eighteen unique sequences were obtained for the PKS ketosynthase (KS) domain of K. brevis. Phylogenetic comparison with closely related PKS genes revealed that 16 grouped with cyanobacteria sequences, while the remaining two grouped with Apicomplexa and previously reported sequences for K. brevis. A fosmid library was also constructed to further characterize PKS genes detected in K. brevis Wilson clone. Several fosmid clones were positive for the presence of PKS genes, and one was fully sequenced to determine the full structure of the PKS cluster. A hybrid non ribosomal peptide synthetase and PKS (NRPS-PKS) gene cluster of 16,061 bp was isolated. In addition, we assessed whether the isolated gene was being actively expressed using reverse transcription polymerase chain reaction (RT-PCR) and determined its localization at the cellular level by chloroplast isolation. RT-PCR analyses revealed that this gene was actively expressed in K. brevis cultures. The hybrid NRPS-PKS gene cluster was located in the chloroplast, suggesting that K. brevis acquired the ability to produce some of its secondary metabolites through endosymbiosis with ancestral cyanobacteria. Further work is needed to determine the compound produced by the NRPS-PKS hybrid, to find other PKS gene sequences, and to assess their role in K. brevis toxin biosynthetic pathway.

  13. Bioinformatics Tools for the Discovery of New Nonribosomal Peptides

    DEFF Research Database (Denmark)

    Leclère, Valérie; Weber, Tilmann; Jacques, Philippe

    2016-01-01

    -dimensional structure of the peptides can be compared with the structural patterns of all known NRPs. The presented workflow leads to an efficient and rapid screening of genomic data generated by high throughput technologies. The exploration of such sequenced genomes may lead to the discovery of new drugs (i......This chapter helps in the use of bioinformatics tools relevant to the discovery of new nonribosomal peptides (NRPs) produced by microorganisms. The strategy described can be applied to draft or fully assembled genome sequences. It relies on the identification of the synthetase genes...

  14. Implementation of communication-mediating domains for non-ribosomal peptide production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Siewers, Verena; San-Bento, Rita; Nielsen, Jens

    2010-01-01

    Saccharomyces cerevisiae has in several cases been proven to be a suitable host for the production of natural products and was recently exploited for the production of non-ribosomal peptides. Synthesis of non-ribosomal peptides (NRPs) is mediated by NRP synthetases (NRPSs), modular enzymes, which...

  15. Portability of oxidase domains in nonribosomal peptide synthetase modules.

    Science.gov (United States)

    Schneider, Tanya L; Walsh, Christopher T

    2004-12-21

    Oxazole and thiazole rings are present in numerous nonribosomal peptide natural products. Oxidase domains are responsible for catalyzing the oxidation of thiazolines and oxazolines to yield fully aromatic heterocycles. Unlike most domains, the placement of oxidase domains within assembly line modules varies. Noting this tolerance, we investigated the portability of an oxidase domain to a heterologous assembly line. The epimerase domain of PchE, involved in pyochelin biosynthesis, was replaced with the oxidase domain from MtaD, involved in myxothiazol biosynthesis. The chimeric module was expressed in soluble form as a flavin mononucleotide-containing flavoprotein. The functionality of the inserted oxidase domain was assayed within PchE and in transfer of the growing siderophore acyl chain from PchE to the next downstream module. While pyochelin-like product release was not observed downstream, the robust activity of the transplanted oxidase domain and the ability of the chimeric module to produce an advanced intermediate bound to the synthetase underscore the possibility of future engineering within nonribosomal peptide synthetase pathways using oxidase domains.

  16. A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria.

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    Chen, Yunqiu; Ntai, Ioanna; Ju, Kou-San; Unger, Michelle; Zamdborg, Leonid; Robinson, Sarah J; Doroghazi, James R; Labeda, David P; Metcalf, William W; Kelleher, Neil L

    2012-01-01

    Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.

  17. Anthranilate-activating modules from fungal nonribosomal peptide assembly lines.

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    Ames, Brian D; Walsh, Christopher T

    2010-04-20

    Fungal natural products containing benzodiazepinone- and quinazolinone-fused ring systems can be assembled by nonribosomal peptide synthetases (NRPS) using the conformationally restricted beta-amino acid anthranilate as one of the key building blocks. We validated that the first module of the acetylaszonalenin synthetase of Neosartorya fischeri NRRL 181 activates anthranilate to anthranilyl-AMP. With this as a starting point, we then used bioinformatic predictions about fungal adenylation domain selectivities to identify and confirm an anthranilate-activating module in the fumiquinazoline A producer Aspergillus fumigatus Af293 as well as a second anthranilate-activating NRPS in N. fischeri. This establishes an anthranilate adenylation domain code for fungal NRPS and should facilitate detection and cloning of gene clusters for benzodiazepine- and quinazoline-containing polycyclic alkaloids with a wide range of biological activities.

  18. Structural insights into nonribosomal peptide enzymatic assembly lines.

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    Koglin, Alexander; Walsh, Christopher T

    2009-08-01

    Nonribosomal peptides have a variety of medicinal activities including activity as antibiotics, antitumor drugs, immunosuppressives, and toxins. Their biosynthesis on multimodular assembly lines as a series of covalently tethered thioesters, in turn covalently attached on pantetheinyl arms on carrier protein way stations, reflects similar chemical logic and protein machinery to fatty acid and polyketide biosynthesis. While structural information on excised or isolated catalytic adenylation (A), condensation (C), peptidyl carrier protein (PCP) and thioesterase (TE) domains had been gathered over the past decade, little was known about how the NRPS catalytic and carrier domains interact with each other both within and across elongation or termination modules. This Highlight reviews recent breakthrough achievements in both X-ray and NMR spectroscopic studies that illuminate the architecture of NRPS PCP domains, PCP-containing didomain-fragments and of a full termination module (C-A-PCP-TE).

  19. Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

    Indian Academy of Sciences (India)

    MANGAL SINGH; SANDEEP CHAUDHARY; DIPTI SAREEN

    2017-03-01

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are themajor multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically importantantibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid,followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, theknowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of theever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thusdeciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is thesubstrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtHhomolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequencedgenome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPSgene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

  20. Diversity of nature's assembly lines - recent discoveries in non-ribosomal peptide synthesis.

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    Payne, Jennifer A E; Schoppet, Melanie; Hansen, Mathias Henning; Cryle, Max J

    2016-12-20

    The biosynthesis of complex natural products by non-ribosomal peptide synthetases (NRPSs) and the related polyketide synthases (PKSs) represents a major source of important bioactive compounds. These large, multi-domain machineries are able to produce a fascinating range of molecules due to the nature of their modular architectures, which allows natural products to be assembled and tailored in a modular, step-wise fashion. In recent years there has been significant progress in characterising the important domains and underlying mechanisms of non-ribosomal peptide synthesis. More significantly, several studies have uncovered important examples of novel activity in many NRPS domains. These discoveries not only greatly increase the structural diversity of the possible products of NRPS machineries but - possibly more importantly - they improve our understanding of what is a highly important, yet complex, biosynthetic apparatus. In this review, several recent examples of novel NRPS function will be introduced, which highlight the range of previously uncharacterised activities that have now been detected in the biosynthesis of important natural products by these mega-enzyme synthetases.

  1. Cyclization of polyketides and non-ribosomal peptides on and off their assembly lines.

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    Pang, Bo; Wang, Min; Liu, Wen

    2016-02-01

    Modular polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are multifunctional megaenzymes that serve as templates to program the assembly of short carboxylic acids and amino acids in a primarily co-linear manner. The variation, combination, permutation and evolution of their functional units (e.g., modules, domains and proteins) along with their association with external enzymes have resulted in the generation of numerous versions of templates, the roles of which have not been fully recognized in the structural diversification of polyketides, non-ribosomal peptides and their hybrids present in nature. In this Highlight, we focus on the assembly-line enzymology and associated chemistry by providing examples of some newly characterized cyclization reactions that occur on and off the assembly lines during and after chain elongation for the purpose of elucidating the template effects of PKSs and NRPSs. A fundamental understanding of the underlying biosynthetic logic would facilitate the elucidation of chemical information contained within the PKS or NRPS templates and benefit the development of strategies for genome mining, biosynthesis-inspired chemical synthesis and combinatorial biosynthesis.

  2. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

    Science.gov (United States)

    Hansen, Frederik T; Gardiner, Donald M; Lysøe, Erik; Fuertes, Patricia Romans; Tudzynski, Bettina; Wiemann, Philipp; Sondergaard, Teis Esben; Giese, Henriette; Brodersen, Ditlev E; Sørensen, Jens Laurids

    2015-02-01

    Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

  3. Quick guide to polyketide synthase and nonribosomal synthetase genes in Fusarium

    DEFF Research Database (Denmark)

    Hansen, Jørgen T.; Sørensen, Jens L.; Giese, Henriette;

    2012-01-01

    Fusarium species produce a plethora of bioactive polyketides and nonribosomal peptides that give rise to health problems in animals and may have drug development potential. Using the genome sequences for Fusarium graminearum, F. oxysporum, F. solani and F. verticillioides we developed a framework...... and NRPS genes in sequenced Fusarium species and their known products. With the rapid increase in the number of sequenced fungal genomes a systematic classification will greatly aid the scientific community in obtaining an overview of the number of different NRPS and PKS genes and their potential...

  4. Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

    Directory of Open Access Journals (Sweden)

    Iyer Lakshminarayan M

    2010-08-01

    Full Text Available Abstract Background Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS in non-ribosomal peptide ligation. Results Here we describe novel examples of AAtRS related proteins that are likely to be involved in the synthesis of widely distributed peptide-derived metabolites. Using sensitive sequence profile methods we show that the cyclodipeptide synthases (CDPSs are members of the HUP class of Rossmannoid domains and are likely to be highly derived versions of the class-I AAtRS catalytic domains. We also identify the first eukaryotic CDPSs in fungi and in animals; they might be involved in immune response in the latter organisms. We also identify a paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptide- derived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS. We further identify an analogous system wherein the MtRS has been replaced by more typical peptide ligases with the ATP-grasp or modular condensation-domains. Conclusions The prevalence of these predicted biosynthetic pathways in phylogenetically distant, pathogenic or symbiotic bacteria suggests that metabolites synthesized by them might participate in interactions with the host. More generally, these findings point to a complete spectrum of recruitment of AAtRS to various non-ribosomal biosynthetic pathways, ranging from the

  5. β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis.

    Science.gov (United States)

    Gaudelli, Nicole M; Long, Darcie H; Townsend, Craig A

    2015-04-16

    Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

  6. Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases.

    Science.gov (United States)

    Bloudoff, Kristjan; Alonzo, Diego A; Schmeing, T Martin

    2016-03-17

    Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.

  7. A Proteomic Survey of Nonribosomal Peptide and Polyketide Biosynthesis in Actinobacteria

    OpenAIRE

    Chen, Yunqiu; Ntai, Ioanna; Ju, Kou-San; Unger, Michelle; Zamdborg, Leonid; Robinson, Sarah J.; Doroghazi, James R.; Labeda, David P.; Metcalf, William W.; Kelleher, Neil L.

    2011-01-01

    Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a “protein-first” method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes ...

  8. Prediction of monomer isomery in Florine: a workflow dedicated to nonribosomal peptide discovery.

    Directory of Open Access Journals (Sweden)

    Thibault Caradec

    Full Text Available Nonribosomal peptides represent a large variety of natural active compounds produced by microorganisms. Due to their specific biosynthesis pathway through large assembly lines called NonRibosomal Peptide Synthetases (NRPSs, they often display complex structures with cycles and branches. Moreover they often contain non proteogenic or modified monomers, such as the D-monomers produced by epimerization. We investigate here some sequence specificities of the condensation (C and epimerization (E domains of NRPS that can be used to predict the possible isomeric state (D or L of each monomer in a putative peptide. We show that C- and E- domains can be divided into 2 sub-regions called Up-Seq and Down-Seq. The Up-Seq region corresponds to an InterPro domain (IPR001242 and is shared by C- and E-domains. The Down-Seq region is specific to the enzymatic activity of the domain. Amino-acid signatures (represented as sequence logos previously described for complete C-and E-domains have been restricted to the Down-Seq region and amplified thanks to additional sequences. Moreover a new Down-Seq signature has been found for Ct-domains found in fungi and responsible for terminal cyclization of the peptides. The identification of these signatures has been included in a workflow named Florine, aimed to predict nonribosomal peptides from NRPS sequence analyses. In some cases, the prediction of isomery is guided by genus-specific rules. Florine was used on a Pseudomonas genome to allow the determination of the type of pyoverdin produced, the update of syringafactin structure and the identification of novel putative products.

  9. SANDPUMA: Ensemble Predictions of Nonribosomal Peptide Chemistry Reveals Biosynthetic Diversity across Actinobacteria.

    Science.gov (United States)

    Chevrette, Marc G; Aicheler, Fabian; Kohlbacher, Oliver; Currie, Cameron R; Medema, Marnix H

    2017-06-19

    Nonribosomally synthesized peptides (NRPs) are natural products with widespread applications in medicine and biotechnology. Many algorithms have been developed to predict the substrate specificities of nonribosomal peptide synthetase adenylation (A) domains from DNA sequences, which enables prioritization and dereplication, and integration with other data types in discovery efforts. However, insufficient training data and a lack of clarity regarding prediction quality have impeded optimal use. Here, we introduce prediCAT, a new phylogenetics-inspired algorithm, which quantitatively estimates the degree of predictability of each A-domain. We then systematically benchmarked all algorithms on a newly-gathered, independent test set of 434 A-domain sequences, showing that active-site-motif-based algorithms outperform whole-domain-based methods. Subsequently, we developed SANDPUMA, a powerful ensemble algorithm, based on newly-trained versions of all high-performing algorithms, which significantly outperforms individual methods. Finally, we deployed SANDPUMA in a systematic investigation of 7,635 Actinobacteria genomes, suggesting that NRP chemical diversity is much higher than previously estimated. SANDPUMA has been integrated into the widely-used antiSMASH biosynthetic gene cluster analysis pipeline and is also available as an open-source, standalone tool. SANDPUMA is freely available at https://bitbucket.org/chevrm/sandpuma and as a docker image at https://hub.docker.com/r/chevrm/sandpuma / under the GNU Public License 3 (GPL3). chevrette@wisc.edu , marnix.medema@wur.nl. Supplementary data are available at Bioinformatics online.

  10. Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis.

    Science.gov (United States)

    Bloudoff, Kristjan; Fage, Christopher D; Marahiel, Mohamed A; Schmeing, T Martin

    2017-01-03

    Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.

  11. Molecular Cross-Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions.

    Science.gov (United States)

    Harden, Bradley J; Frueh, Dominique P

    2017-01-24

    Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.

  12. Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

    Directory of Open Access Journals (Sweden)

    Ute Hentschel

    2012-05-01

    Full Text Available Genomic mining revealed one major nonribosomal peptide synthetase (NRPS phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.

  13. Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsu-Hua; Chiang, Yi Ming; Entwistle, Ruth; Ahuja, Mammeet; Lee, Kuan-Han; Bruno, Kenneth S.; Wu, Tung-Kung; Oakley, Berl R.; Wang, Clay C.

    2012-04-10

    Genome sequencing of Aspergillus species including A. nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites have not yet been elucidated. The A. nidulans genome contains twelve nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), and fourteen NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in A. niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

  14. A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

    Directory of Open Access Journals (Sweden)

    Nederbragt Alexander J

    2009-08-01

    Full Text Available Abstract Background Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS. Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454" and mass spectrometry screening of oligopeptides produced in the strain Planktothrix rubescens NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides. Results Thirteen types of oligopeptides were uncovered by mass spectrometry (MS analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded precursor peptide sequences to microviridin and oscillatorin were found in the genes mdnA and oscA, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island. Conclusion Altogether seven nonribosomal peptide synthetase (NRPS gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully

  15. The Role of a Nonribosomal Peptide Synthetase in l-Lysine Lactamization During Capuramycin Biosynthesis.

    Science.gov (United States)

    Liu, Xiaodong; Jin, Yuanyuan; Cui, Zheng; Nonaka, Koichi; Baba, Satoshi; Funabashi, Masanori; Yang, Zhaoyong; Van Lanen, Steven G

    2016-05-03

    Capuramycins are one of several known classes of natural products that contain an l-Lys-derived l-α-amino-ɛ-caprolactam (l-ACL) unit. The α-amino group of l-ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP-independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP-dependent amide-bond-forming catalyst responsible for the biosynthesis of the remaining amide bond present in l-ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l-Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS-mediated mechanism is employed in the biosynthesis of other l-ACL-containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS-derived peptides that contain an unmodified l-Lys residue.

  16. Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

    Directory of Open Access Journals (Sweden)

    Niran Roongsawang

    2010-12-01

    Full Text Available Lipopeptide biosurfactants (LPBSs consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive and Pseudomonas (Gram-negative have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs. Production of active‑form NRPSs requires not only transcriptional induction and translation but also post‑translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

  17. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product...... of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased...... sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P

  18. The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins.

    Science.gov (United States)

    Lohman, Jeremy R; Ma, Ming; Cuff, Marianne E; Bigelow, Lance; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2014-07-01

    Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.

  19. Bioactivities by a crude extract from the Greenlandic Pseudomonas sp. In5 involves the nonribosomal peptides, nunamycin and nunapeptin

    DEFF Research Database (Denmark)

    Frydenlund Michelsen, Charlotte; Jensen, Helle; Venditto, Vincent J.;

    2015-01-01

    Bioactive microbial metabolites provide a successful source of novel compounds with pharmaceutical potentials. The bacterium Pseudomonas sp. In5 is a biocontrol strain isolated from a plant disease suppressive soil in Greenland, which produces two antimicrobial nonribosomal peptides (NRPs......), nunapeptin and nunamycin. In this study, we used in vitro antimicrobial and anticancer bioassays to evaluate the potential bioactivities of both a crude extract derived from Pseudomonas sp. In5 and NRPs purified from the crude extract....

  20. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae.

    Science.gov (United States)

    Siewers, Verena; Chen, Xiao; Huang, Le; Zhang, Jie; Nielsen, Jens

    2009-11-01

    Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5' end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 degrees C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast.

  1. Optimization of nonribosomal peptides production by a psychrotrophic fungus: Trichoderma velutinum ACR-P1.

    Science.gov (United States)

    Sharma, Richa; Singh, Varun P; Singh, Deepika; Yusuf, Farnaz; Kumar, Anil; Vishwakarma, Ram A; Chaubey, Asha

    2016-11-01

    Trichoderma is an anamorphic filamentous fungal genus with immense potential for production of small valuable secondary metabolites with indispensable biological activities. Microbial dynamics of a psychrotrophic strain Trichoderma velutinum ACR-P1, isolated from unexplored niches of the Shiwalik region, bestowed with rich biodiversity of microflora, was investigated for production of nonribosomal peptides (NRPs) by metabolite profiling by intact-cell mass spectrometry (ICMS) employing matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. Being the first report on NRPs production by T. velutinum, studies on optimization of growth conditions by Response Surface Methodology (RSM) for production of NRPs by ACR-P1 was carried out strategically. Multifold enhancement in the yield of NRPs belonging to subfamily SF4 with medium chain of amino acid residues having m/z 1437.9, 1453.9, and 1452.0 at pH 5.9 at 20 °C and of subfamily SF1 with long-chain amino acid residues having m/z 1770.2, 1784.2, 1800.1, 1802.1, and 1815.1 was achieved at pH 7.0 at 25 °C. Complexities of natural mixtures were thus considerably reduced under respective optimized culture conditions accelerating the production of novel microbial natural products by saving time and resources.

  2. Stereochemistry and conformation of skyllamycin, a non-ribosomally synthesized peptide from Streptomyces sp. Acta 2897.

    Science.gov (United States)

    Schubert, Vivien; Di Meo, Florent; Saaidi, Pierre-Loïc; Bartoschek, Stefan; Fiedler, Hans-Peter; Trouillas, Patrick; Süssmuth, Roderich D

    2014-04-22

    Skyllamycin is a non-ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF-signaling. The peptide scaffold contains an N-terminal cinnamoyl moiety, a β-methylation of aspartic acid, three β-hydroxylated amino acids and one rarely occurring α-hydroxy glycine. With the exception of α-hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC-MS and Marfey-HPLC analysis. The stereochemistry of α-hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY-NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R- and S-configured α-hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S-configured α-hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two-component flavin dependent monooxygenase (Sky39) as S-selective.

  3. Photooxidation of the Antimicrobial, Nonribosomal Peptide Bacitracin A by Singlet Oxygen under Environmentally Relevant Conditions.

    Science.gov (United States)

    Lundeen, Rachel A; Chu, Chiheng; Sander, Michael; McNeill, Kristopher

    2016-08-16

    Bacitracin is a mixture of nonribosomal peptides (NRPs) that is extensively used as an antibiotic in both human and veterinary medicine. Despite its widespread use over the past six decades, very few studies have addressed the environmental fate of bacitracin and zinc-bacitracin complexes. In this study, the photochemical transformation of bacitracin components (i.e., cyclic dodecapeptides) in the aquatic environment was investigated. A high resolution mass spectrometry (HRMS)-based approach enabled monitoring of the photochemical degradation kinetics of individual bacitracin components, investigation of the relative contribution of reactive oxygen species (e.g., singlet oxygen, (1)O2) in dissolved organic matter-sensitized photoreactions, and identification of oxidative modifications in bacitracin photoproducts. The results of this study support the hypothesis that indirect photochemical oxidation of the histidine (His) residue by (1)O2 is a major degradation pathway for bacitracin A, the most potent congener of the mixture. Furthermore, the photooxidation rate of bacitracin A with (1)O2 decreased upon bacitracin A coordination with Zn(2+), demonstrating that the photochemistry of metal-bound His is different from that of metal-free His. Overall, these results provide insight into the fate of bacitracin components in the aquatic environment and highlight the potential of utilizing this HRMS-based methodology to study transformations of other environmentally relevant NRPs.

  4. Nonribosomal peptides and polyketides of Burkholderia: new compounds potentially implicated in biocontrol and pharmaceuticals.

    Science.gov (United States)

    Esmaeel, Qassim; Pupin, Maude; Jacques, Philippe; Leclère, Valérie

    2017-05-25

    Bacteria belonging to the genus Burkholderia live in various ecological niches and present a significant role in the environments through the excretion of a wide variety of secondary metabolites including modular nonribosomal peptides (NRPs) and polyketides (PKs). These metabolites represent a widely distributed biomedically and biocontrol important class of natural products including antibiotics, siderophores, and anticancers as well as biopesticides that are considered as a novel source that can be used to defend ecological niche from competitors and to promote plant growth. The aim of this review is to present all NRPs produced or potentially produced by strains of Burkholderia, as NRPs represent a major source of active compounds implicated in biocontrol. The review is a compilation of results from a large screening we have performed on 48 complete sequenced genomes available in NCBI to identify NRPS gene clusters, and data found in the literature mainly because some interesting compounds are produced by strains not yet sequenced. In addition to NRPs, hybrids NRPs/PKs are also included. Specific features about biosynthetic gene clusters and structures of the modular enzymes responsible for the synthesis, the biological activities, and the potential uses in agriculture and pharmaceutical of NRPs and hybrids NRPs/PKs will also be discussed.

  5. Norine, the knowledgebase dedicated to non-ribosomal peptides, is now open to crowdsourcing.

    Science.gov (United States)

    Flissi, Areski; Dufresne, Yoann; Michalik, Juraj; Tonon, Laurie; Janot, Stéphane; Noé, Laurent; Jacques, Philippe; Leclère, Valérie; Pupin, Maude

    2016-01-01

    Since its creation in 2006, Norine remains the unique knowledgebase dedicated to non-ribosomal peptides (NRPs). These secondary metabolites, produced by bacteria and fungi, harbor diverse interesting biological activities (such as antibiotic, antitumor, siderophore or surfactant) directly related to the diversity of their structures. The Norine team goal is to collect the NRPs and provide tools to analyze them efficiently. We have developed a user-friendly interface and dedicated tools to provide a complete bioinformatics platform. The knowledgebase gathers abundant and valuable annotations on more than 1100 NRPs. To increase the quantity of described NRPs and improve the quality of associated annotations, we are now opening Norine to crowdsourcing. We believe that contributors from the scientific community are the best experts to annotate the NRPs they work on. We have developed MyNorine to facilitate the submission of new NRPs or modifications of stored ones. This article presents MyNorine and other novelties of Norine interface released since the first publication. Norine is freely accessible from the following URL: http://bioinfo.lifl.fr/NRP.

  6. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  7. Kinetics profiling of gramicidin S synthetase A, a member of nonribosomal peptide synthetases.

    Science.gov (United States)

    Sun, Xun; Li, Hao; Alfermann, Jonas; Mootz, Henning D; Yang, Haw

    2014-12-23

    Nonribosomal peptide synthetases (NRPS) incorporate assorted amino acid substrates into complex natural products. The substrate is activated via the formation of a reactive aminoacyl adenylate and is subsequently attached to the protein template via a thioester bond. The reactive nature of such intermediates, however, leads to side reactions that also break down the high-energy anhydride bond. The off-pathway kinetics or their relative weights compared to that of the on-pathway counterpart remains generally elusive. Here, we introduce multiplatform kinetics profiling to quantify the relative weights of on- and off-pathway reactions. Using the well-defined stoichiometry of thioester formation, we integrate a mass spectrometry (MS) kinetics assay, a high-performance liquid chromatography (HPLC) assay, and an ATP-pyrophosphate (PPi) exchange assay to map out a highly efficient on-pathway kinetics profile of the substrate activation and intermediate uploading (>98% relative weight) for wide-type gramicidin S synthetase A (GrsA) and a 87% rate profile for a cysteine-free GrsA mutant. Our kinetics profiling approach complements the existing enzyme-coupled byproduct-release assays, unraveling new mechanistic insights of substrate activation/channeling in NRPS enzymes.

  8. Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a Greenlandic suppressive soil.

    Science.gov (United States)

    Michelsen, Charlotte F; Watrous, Jeramie; Glaring, Mikkel A; Kersten, Roland; Koyama, Nobuhiro; Dorrestein, Pieter C; Stougaard, Peter

    2015-03-17

    Potatoes are cultivated in southwest Greenland without the use of pesticides and with limited crop rotation. Despite the fact that plant-pathogenic fungi are present, no severe-disease outbreaks have yet been observed. In this report, we document that a potato soil at Inneruulalik in southern Greenland is suppressive against Rhizoctonia solani Ag3 and uncover the suppressive antifungal mechanism of a highly potent biocontrol bacterium, Pseudomonas fluorescens In5, isolated from the suppressive potato soil. A combination of molecular genetics, genomics, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) revealed an antifungal genomic island in P. fluorescens In5 encoding two nonribosomal peptides, nunamycin and nunapeptin, which are key components for the biocontrol activity by strain In5 in vitro and in soil microcosm experiments. Furthermore, complex microbial behaviors were highlighted. Whereas nunamycin was demonstrated to inhibit the mycelial growth of R. solani Ag3, but not that of Pythium aphanidermatum, nunapeptin instead inhibited P. aphanidermatum but not R. solani Ag3. Moreover, the synthesis of nunamycin by P. fluorescens In5 was inhibited in the presence of P. aphanidermatum. Further characterization of the two peptides revealed nunamycin to be a monochlorinated 9-amino-acid cyclic lipopeptide with similarity to members of the syringomycin group, whereas nunapeptin was a 22-amino-acid cyclic lipopeptide with similarity to corpeptin and syringopeptin. Crop rotation and systematic pest management are used to only a limited extent in Greenlandic potato farming. Nonetheless, although plant-pathogenic fungi are present in the soil, the farmers do not experience major plant disease outbreaks. Here, we show that a Greenlandic potato soil is suppressive against Rhizoctonia solani, and we unravel the key biocontrol components for Pseudomonas fluorescens In5, one of the potent biocontrol bacteria

  9. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles.

    Science.gov (United States)

    Piel, Jörn

    2002-10-29

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were cloned from total DNA of Paederus fuscipes beetles, which use this compound for chemical defense. Sequence analysis of the gene cluster and adjacent regions revealed the presence of ORFs with typical bacterial architecture and homologies. The ped cluster, which is present only in beetle specimens with high pederin content, is located on a 54-kb region bordered by transposase pseudogenes and encodes a mixed modular polyketide synthase/nonribosomal peptide synthetase. Notably, none of the modules contains regions with homology to acyltransferase domains, but two copies of isolated monodomain acyltransferase genes were found at the upstream end of the cluster. In line with an involvement in pederin biosynthesis, the upstream cluster region perfectly mirrors pederin structure. The unexpected presence of additional polyketide synthase/nonribosomal peptide synthetase modules reveals surprising insights into the evolutionary relationship between pederin-type pathways in beetles and sponges.

  10. Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis.

    Science.gov (United States)

    Yeh, Hsu-Hua; Chiang, Yi-Ming; Entwistle, Ruth; Ahuja, Manmeet; Lee, Kuan-Han; Bruno, Kenneth S; Wu, Tung-Kung; Oakley, Berl R; Wang, Clay C C

    2012-11-01

    Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

  11. The Stress-responsive and Host-oriented Role of Nonribosomal Peptide Synthetases in an Entomopathogenic Fungus, Beauveria bassiana.

    Science.gov (United States)

    Liu, Hang; Xie, Linan; Wang, Jing; Guo, Qiannan; Yang, Shengnan; Liang, Pei; Wang, Chengshu; Lin, Min; Xu, Yuquan; Zhang, Liwen

    2016-11-14

    Beauveria bassiana infects numbers of pest species and is known to produce insecticidal substances, e.g., the nonribosomal peptides (NRPs) beauvericin and bassianolide. However, most NRPs and their biological roles in B. bassiana remain undiscovered. To identify NRPs that potentially contribute to pathogenesis, the 21 predicted NRP synthetases (NRPSs) or NRPS-like proteins of B. bassiana ARSEF2860 were primarily ranked into three functional groups: basic metabolism (7 NRPSs), pathogenicity (12 NRPSs) and unknown function (2 NRPSs). Based on the transcript levels during in vivo growth on diamondback moth (Plutella xylostella, Linnaeus), half of the Group II NRPSs were likely to be involved in infection. Given that the metabolites biosynthesized by these NRPSs remaining to determined, our result underlines the importance of NRPSome in fungal pathogenesis, and will serve as a guide for future genomic mining projects to discover functionally essential and structurally diverse NRPs in fungal genomes.

  12. Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus.

    Science.gov (United States)

    O'Hanlon, Karen A; Gallagher, Lorna; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas O; Doyle, Sean

    2012-05-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H(2)O(2), and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

  13. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M.

    2016-09-05

    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

  14. Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces.

    Science.gov (United States)

    O'Connor, Sarah E; Chen, Huawei; Walsh, Christopher T

    2002-04-30

    The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.

  15. New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

    Energy Technology Data Exchange (ETDEWEB)

    Gallo, A.; Bruno, K. S.; Solfrizzo, M.; Perrone, G.; Mule, G.; Visconti, A.; Baker, S. E.

    2012-09-14

    Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been obtained in Penicillium species. In Aspergillus species only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase in OTA producing A. carbonarius ITEM 5010 has removed the ability of the fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in Aspergillus species. The absence of OTA and ochratoxin α-the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β- the dechloro analog of ochratoxin α- were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius, and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight in the biosynthetic pathway of the toxin.

  16. The long-overlooked enzymology of a nonribosomal peptide synthetase-independent pathway for virulence-conferring siderophore biosynthesis.

    Science.gov (United States)

    Oves-Costales, Daniel; Kadi, Nadia; Challis, Gregory L

    2009-11-21

    Siderophores are high-affinity ferric iron chelators biosynthesised and excreted by most microorganisms that play an important role in iron acquisition. Siderophore-mediated scavenging of ferric iron from hosts contributes significantly to the virulence of pathogenic microbes. As a consequence siderophore biosynthesis is an attractive target for chemotherapeutic intervention. Two main pathways for siderophore biosynthesis exist in microbes. One pathway involves nonribosomal peptide synthetase (NRPS) multienzymes while the other is NRPS-independent. The enzymology of NRPS-mediated siderophore biosynthesis has been extensively studied for more than a decade. In contrast, the enzymology of NRPS-independent siderophore (NIS) biosynthesis was overlooked for almost thirty years since the first genetic characterisation of the NIS biosynthetic pathway to aerobactin. However, the past three years have witnessed an explosion of interest in the enzymology of NIS synthetases, the key enzymes in the assembly of siderophores via the NIS pathway. The biochemical characterisation of ten purified recombinant synthetases has been reported since 2007, along with the first structural characterisation of a synthetase by X-ray crystallography in 2009. In this feature article we summarise the recent progress that has been made in understanding the long-overlooked enzymology of NRPS-independent siderophore biosynthesis, highlight important remaining questions, and suggest likely directions for future research.

  17. Rational design of a bimodular model system for the investigation of heterocyclization in nonribosomal peptide biosynthesis.

    Science.gov (United States)

    Duerfahrt, Thomas; Eppelmann, Katrin; Müller, Rolf; Marahiel, Mohamed A

    2004-02-01

    Cyclization (Cy) domains in NRPS catalyze the heterocyclization of cysteine and serine/threonine to thiazoline and oxazoline rings. A model system consisting of the first two modules of bacitracin synthetase A fused to the thioesterase (Te) domain of tyrocidine synthetase was constructed (BacA1-2-Te) and shown to be active in production of the heterocyclic IleCys(thiazoline). Based on this model system, the feasibility of Cy domain module fusions was investigated by replacing the BacA2 Cy-A-PCP-module with modules of MbtB and MtaD from the biosynthesis systems of mycobactin and myxothiazol, revealing the formation of novel heterocyclic dipeptides. To dissect the reaction sequence of the Cy domain in peptide bond formation and heterocyclization, several residues of the BacA1-2-Te Cy domain were analyzed by mutagenesis. Two mutants exhibited formation of the noncyclic dipeptide, providing clear evidence for the independence of condensation and cyclization.

  18. Discovery of antibacterials and other bioactive compounds from microorganisms-evaluating methodologies for discovery and generation of non-ribosomal peptide antibiotics.

    Science.gov (United States)

    Witting, K; Süssmuth, R D

    2011-10-01

    After decades of neglect in industrial research the comeback of natural products is due since improved screening approaches are at disposal, yielding a multitude of new compounds from natural sources. Besides traditional compound libraries peptides are characterized by an enormous structural complexity, thus increasing the chance of finding a hit in a screening. Emphasizing antibacterial compounds structural complexity is a prerequisite for their success. This review focuses on the screening approaches employed for the discovery of mostly antibacterial, non-ribosomal peptides derived from natural sources. Traditional screening methodologies as well as genetic approaches are discussed in this context. Utilizing genetic engineering methods e.g., precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis, as well as chemoenzymatics to achieve greater structural diversity is thoroughly discussed and exemplified by recent discoveries.

  19. Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

    OpenAIRE

    2010-01-01

    Previously melittin, the α-helical basic honey bee venom peptide, was shown to inhibit F1-ATPase by binding at the β-subunit DELSEED motif of F1Fo ATP synthase. Herein, we present the inhibitory effects of the basic α-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F1 and membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by amphib...

  20. Surveys of non-ribosomal peptide and polyketide assembly lines in fungi and prospects for their analysis in vitro and in vivo.

    Science.gov (United States)

    Evans, Bradley S; Robinson, Sarah J; Kelleher, Neil L

    2011-01-01

    With many bioactive non-ribosomal peptides and polyketides produced in fungi, studies of their biosyntheses are an active area of research. Practical limitations of working with mega-dalton synthetases including cell lysis and protein extraction to recombinant gene and pathway expression has slowed understanding of many secondary metabolic processes relative to bacterial counterparts. Recent advances in accessing fungal biosynthetic machinery are beginning to change this. Here we describe the successes of some studies of thiotemplate biosynthesis in fungal systems, along with very recent advances in chemical tagging and mass spectrometric strategies to selectively study biosynthetic conveyer belts in isolation, and within a few years, in endogenous fungal proteomes. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. A sensitive single-enzyme assay system using the non-ribosomal peptide synthetase BpsA for measurement of L-glutamine in biological samples

    Science.gov (United States)

    Brown, Alistair S.; Robins, Katherine J.; Ackerley, David F.

    2017-01-01

    The ability to rapidly, economically and accurately measure L-glutamine concentrations in biological samples is important for many areas of research, medicine or industry, however there is room for improvement on existing methods. We describe here how the enzyme BpsA, a single-module non-ribosomal peptide synthetase able to convert L-glutamine into the blue pigment indigoidine, can be used to accurately measure L-glutamine in biological samples. Although indigoidine has low solubility in aqueous solutions, meaning direct measurements of indigoidine synthesis do not reliably yield linear standard curves, we demonstrate that resolubilisation of the reaction end-products in DMSO overcomes this issue and that spontaneous reduction to colourless leuco-indigoidine occurs too slowly to interfere with assay accuracy. Our protocol is amenable to a 96-well microtitre format and can be used to measure L-glutamine in common bacterial and mammalian culture media, urine, and deproteinated plasma. We show that active BpsA can be prepared in high yield by expressing it in the apo-form to avoid the toxicity of indigoidine to Escherichia coli host cells, then activating it to the holo-form in cell lysates prior to purification; and that BpsA has a lengthy shelf-life, retaining >95% activity when stored at either −20 °C or 4 °C for 24 weeks. PMID:28139746

  2. Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani.

    Science.gov (United States)

    Romans-Fuertes, Patricia; Sondergaard, Teis Esben; Sandmann, Manuela Ilse Helga; Wollenberg, Rasmus Dam; Nielsen, Kristian Fog; Hansen, Frederik T; Giese, Henriette; Brodersen, Ditlev Egeskov; Sørensen, Jens Laurids

    2016-11-01

    Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.

  3. Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes.

    Science.gov (United States)

    Bakal, Tomas; Goo, Kian-Sim; Najmanova, Lucie; Plhackova, Kamila; Kadlcik, Stanislav; Ulanova, Dana

    2015-11-01

    In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.

  4. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Science.gov (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  5. Phytochelatin synthase: of a protease a peptide polymerase made.

    Science.gov (United States)

    Rea, Philip A

    2012-05-01

    Of the mechanisms known to protect vascular plants and some algae, fungi and invertebrates from the toxic effects of non-essential heavy metals such as As, Cd or Hg, one of the most sophisticated is the enzyme-catalyzed synthesis of phytochelatins (PCs). PCs, (γ-Glu-Cys)(n) Gly polymers, which serve as high-affinity, thiol-rich cellular chelators and contribute to the detoxification of heavy metal ions, are derived from glutathione (GSH; γ-Glu-Cys-Gly) and related thiols in a reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15). Using the enzyme from Arabidopsis thaliana (AtPCS1) as a model, the reasoning and experiments behind the conclusion that PC synthases are novel papain-like Cys protease superfamily members are presented. The status of S-substituted GSH derivatives as generic PC synthase substrates and the sufficiency of the N-terminal domain of the enzyme from eukaryotic and its half-size equivalents from prokaryotic sources, for net PC synthesis and deglycylation of GSH and its derivatives, respectively, are emphasized. The question of the common need or needs met by PC synthases and their homologs is discussed. Of the schemes proposed to account for the combined protease and peptide polymerase capabilities of the eukaryotic enzymes vs the limited protease capabilities of the prokaryotic enzymes, two that will be considered are the storage and homeostasis of essential heavy metals in eukaryotes and the metabolism of S-substituted GSH derivatives in both eukaryotes and prokaryotes.

  6. From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase.

    Science.gov (United States)

    Li, Y M; Milne, J C; Madison, L L; Kolter, R; Walsh, C T

    1996-11-15

    Esherichia coli microcin B17 is a posttranslationally modified peptide that inhibits bacterial DNA gyrase. It contains four oxazole and four thiazole rings and is representative of a broad class of pharmaceutically important natural products with five-membered heterocycles derived from peptide precursors. An in vitro assay was developed to detect heterocycle formation, and an enzyme complex, microcin B17 synthase, was purified and found to contain three proteins, McbB, McbC, and McbD, that convert 14 residues into the eight mono- and bisheterocyclic moieties in vitro that confer antibiotic activity on mature microcin B17. These enzymatic reactions alter the peptide backbone connectivity. The propeptide region of premicrocin is the major recognition determinant for binding and downstream heterocycle formation by microcin B17 synthase. A general pathway for the enzymatic biosynthesis of these heterocycles is formulated.

  7. A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

    Directory of Open Access Journals (Sweden)

    Jan Mareš

    Full Text Available A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea. Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

  8. Asp residues of βDELSEED-motif are required for peptide binding in the Escherichia coli ATP synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Tayou, Junior; Laughlin, Thomas F

    2015-04-01

    This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Elevation in sphingomyelin synthase activity is associated with increases in amyloid-beta peptide generation.

    Directory of Open Access Journals (Sweden)

    Jen-Hsiang T Hsiao

    Full Text Available A pathological hallmark of Alzheimer's disease (AD is the presence of amyloid-beta peptide (Aβ plaques in the brain. Aβ is derived from a sequential proteolysis of the transmenbrane amyloid precursor protein (APP, a process which is dependent on the distribution of lipids present in the plasma membrane. Sphingomyelin is a major membrane lipid, however its role in APP processing is unclear. Here, we assessed the expression of sphingomyelin synthase (SGMS1; the gene responsible for sphingomyelin synthesis in human brain and found that it was significantly elevated in the hippocampus of AD brains, but not in the cerebellum. Secondly, we assessed the impact of altering SGMS activity on Aβ generation. Inhibition of SGMS activity significantly reduced the level of Aβ in a dose- and time dependent manner. The decrease in Aβ level occurred without changes in APP expression or cell viability. These results when put together indicate that SGMS activity impacts on APP processing to produce Aβ and it could be a contributing factor in Aβ pathology associated with AD.

  10. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  11. In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms.

    Science.gov (United States)

    Marani, Mariela M; Costa, Joana; Mafra, Isabel; Oliveira, Maria Beatriz P P; Camperi, Silvia A; Leite, José Roberto de Souza Almeida

    2015-03-01

    For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection.

  12. Appetite suppressive effects of yeast hydrolysate on nitric oxide synthase (NOS) expression and vasoactive intestinal peptide (VIP) immunoreactivity in hypothalamus.

    Science.gov (United States)

    Jung, E Y; Suh, H J; Kim, S Y; Hong, Y S; Kim, M J; Chang, U J

    2008-11-01

    To investigate the effects of yeast hydrolysate on appetite regulation mechanisms in the central nervous system, nitric oxide synthase (NOS) expression and vasoactive intestinal peptide (VIP) immunoreactivity in the paraventricular nucleus (PVN) and ventromedial hypothalamic nucleus (VMH) of the hypothalamus were examined. Male Sprague-Dawley (SD) rats were assigned to five groups: control (normal diet), BY-1 and BY-2 (normal diet with oral administration of 0.1 g and 1.0 g of yeast hydrolysate yeast hydrolysate 10-30 kDa/kg body weight, respectively). The body weight gain in the BY groups was less than that in the control. In particular, the weight gain of the BY-2 group (133.0 +/- 5.1 g) was significantly lower (p yeast hydrolysate of <10 kDa reduced the body weight gain and body fat in normal diet-fed rats and increased the lipid energy metabolism by altering the expression of NOS and VIP in neurons.

  13. Vascular relaxation induced by C-type natriuretic peptide involves the ca2+/NO-synthase/NO pathway.

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    Fernanda A Andrade

    Full Text Available AIMS: C-type natriuretic peptide (CNP and nitric oxide (NO are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3 contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery. MAIN METHODS: Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry. KEY FINDINGS: CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas. SIGNIFICANCE: These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.

  14. Etiology of Alzheimer's disease: kinetic, thermodynamic and fluorimetric analyses of interactions of pseudo Aβ-peptides with neuronal nitric oxide synthase.

    Science.gov (United States)

    Padayachee, E R; Whiteley, C G

    2013-10-01

    Aggregated β-amyloid deposit is a hallmark in the neuropathology of Alzheimer's disease but their mechanism of formation still remains unresolved. Previously we reported that a normal pentapeptide Aβ(17-21) and glycine zipper peptide Aβ(29-33) strongly inhibited nitric oxide synthase and rapidly initiated fibrillogenesis. Critical amino acids within these fragments were not identified. We now report on the interaction of four pseudo-peptides with nNOS - two peptides with a reversed amino acid sequence [Aβ(17-21r); Aβ(29-33r)] and two peptides with Phe19, Phe20 and Ile31, Ile32 substituted with polar glutamic acid [Aβ(17-21p); Aβ(29-33p)]. It was shown that while the inhibitor constants (Ki) increased 2-3 fold for each of the pseudo-peptides when compared with the normal peptides the dissociation constant Kd increased between 20 and 50 fold. Stern-Volmer fluorescence quenching constants (K(SV)) for Aβ(17-21p) and Aβ(29-33p) were 7.2×10(-3) and 6.1×10(-3) μM(-1) respectively at 298 K some 2-3 fold lower than the corresponding Aβ(17-21r); Aβ(29-33r). With temperature increase there was an increase in K(SV) and Kd, suggesting a dynamic quenching mechanism. Thermodynamic parameters, ΔH, ΔS and ΔG were all positive indicating endothermic, non-spontaneous, hydrophobic-hydrophobic associations of the pseudo-peptides with the enzyme. By FRET analysis the efficiency of fluorescence transfer between enzyme tryptophans and the pseudo-peptides was 90% (compared to 97% for the natural substrate). The distance the tryptophans moved after interaction with Aβ(17-21r) and Aβ(17-21p) was 10% greater, while for Aβ(29-33r) and Aβ(29-33p) it was 20-25% greater, than with the normal peptides; the fluorescence intensity was 20-75% higher. This increase in distance, fluorescent intensity and transfer efficiency illustrate an increase in interaction energy for the pseudo-peptides with nNOS lending support for the strategic position of the Phe19, Phe20, Ile31 and Ile32

  15. Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain-domain communication and allosteric regulation.

    Science.gov (United States)

    Landau, Mark J; Sharma, Hitesh; Anderson, Karen S

    2013-09-01

    The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking β-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these β-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance. © 2013 The Protein Society.

  16. Changes in cardiac aldosterone and its synthase in rats with chronic heart failure: an intervention study of long-term treatment with recombinant human brain natriuretic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, X.Q. [Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Department of Cardiology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, Hubei (China); Hong, H.S. [Department of Geriatrics, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Lin, X.H. [Department of Emergency Medicine, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Chen, L.L. [Department of Cardiology, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Li, Y.H. [Department of Cardiology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, Hubei (China)

    2014-07-11

    The physiological mechanisms involved in isoproterenol (ISO)-induced chronic heart failure (CHF) are not fully understood. In this study, we investigated local changes in cardiac aldosterone and its synthase in rats with ISO-induced CHF, and evaluated the effects of treatment with recombinant human brain natriuretic peptide (rhBNP). Sprague-Dawley rats were divided into 4 different groups. Fifty rats received subcutaneous ISO injections to induce CHF and the control group (n=10) received equal volumes of saline. After establishing the rat model, 9 CHF rats received no further treatment, rats in the low-dose group (n=8) received 22.5 μg/kg rhBNP and those in the high-dose group (n=8) received 45 μg/kg rhBNP daily for 1 month. Cardiac function was assessed by echocardiographic and hemodynamic analysis. Collagen volume fraction (CVF) was determined. Plasma and myocardial aldosterone concentrations were determined using radioimmunoassay. Myocardial aldosterone synthase (CYP11B2) was detected by quantitative real-time PCR. Cardiac function was significantly lower in the CHF group than in the control group (P<0.01), whereas CVF, plasma and myocardial aldosterone, and CYP11B2 transcription were significantly higher than in the control group (P<0.05). Low and high doses of rhBNP significantly improved hemodynamics (P<0.01) and cardiac function (P<0.05) and reduced CVF, plasma and myocardial aldosterone, and CYP11B2 transcription (P<0.05). There were no significant differences between the rhBNP dose groups (P>0.05). Elevated cardiac aldosterone and upregulation of aldosterone synthase expression were detected in rats with ISO-induced CHF. Administration of rhBNP improved hemodynamics and ventricular remodeling and reduced myocardial fibrosis, possibly by downregulating CYP11B2 transcription and reducing myocardial aldosterone synthesis.

  17. Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase).

    Science.gov (United States)

    Perrine, Cynthia; Ju, Tongzhong; Cummings, Richard D; Gerken, Thomas A

    2009-03-01

    Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood; however, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison with the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg, and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests that there are indeed subtle specific and nonspecific protein-targeting sequence motifs for this transferase.

  18. Deletion of the RluD pseudouridine synthase promotes SsrA peptide tagging of ribosomal protein S7.

    Science.gov (United States)

    Schaub, Ryan E; Hayes, Christopher S

    2011-01-01

    RluD catalyses formation of three pseudouridine residues within helix 69 of the 50S ribosome subunit. Helix 69 makes important contacts with the decoding centre on the 30S subunit and deletion of rluD was recently shown to interfere with translation termination in Escherichia coli. Here, we show that deletion of rluD increases tmRNA activity on ribosomes undergoing release factor 2 (RF2)-mediated termination at UGA stop codons. Strikingly, tmRNA-mediated SsrA peptide tagging of two proteins, ribosomal protein S7 and LacI, was dramatically increased in ΔrluD cells. S7 tagging was due to a unique C-terminal peptide extension found in E. coli K-12 strains. Introduction of the rpsG gene (encoding S7) from an E. coli B strain abrogated S7 tagging in the ΔrluD background, and partially complemented the mutant's slow-growth phenotype. Additionally, exchange of the K-12 prfB gene (encoding RF2) with the B strain allele greatly reduced tagging in ΔrluD cells. In contrast to E. coli K-12 cells, deletion of rluD in an E. coli B strain resulted in no growth phenotype. These findings indicate that the originally observed rluD phenotypes result from synthetic interactions with rpsG and prfB alleles found within E. coli K-12 strains.

  19. Structure-based design of a potent and selective small peptide inhibitor of Mycobacterium tuberculosis 6-hydroxymethyl-7, 8-dihydropteroate synthase: a computer modelling approach.

    Science.gov (United States)

    Rao, Gita Subba; Kumar, Manoj

    2008-06-01

    In an attempt to design novel anti-TB drugs, the target chosen is the enzyme 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS), which is an attractive target since it is present in microorganisms but not in humans. The existing drugs for this target are the sulfa drugs, which have been used for about seven decades. However, single mutations in the DHPS gene can cause resistance to sulfa drugs. Therefore, there is a need for the design of novel drugs. Based on the recently determined crystal structure of Mycobacterium tuberculosis (M.tb) DHPS complexed with a known substrate analogue, and on the crystal structures of E. coli DHPS and Staphylococcus aureus DHPS, we have identified a dipeptide inhibitor with the sequence WK. Docking calculations indicate that this peptide has a significantly higher potency than the sulfa drugs. In addition, the potency is 70-90 times higher for M.tb DHPS as compared to that for the pterin and folate-binding sites of key human proteins. Thus, the designed inhibitor is a promising lead compound for the development of novel antimycobcaterial agents.

  20. Neuronal nitric oxide synthase immunoreactivity in the guinea-pig liver: distribution and colocalization with neuropeptide Y and calcitonin gene-related peptide.

    Science.gov (United States)

    Esteban, F J; Jiménez, A; Fernández, A P; del Moral, M L; Sánchez-López, A M; Hernández, R; Garrosa, M; Pedrosa, J A; Rodrigo, J; Peinado, M A

    2001-12-01

    The innervation pattern of the guinea-pig liver is similar to that of the human liver. However, many aspects of the distribution of the neuronal isoform of the enzyme nitric oxide synthase (nNOS) in the guinea-pig liver and its colocalization with neuropeptides remain to be elucidated. The distribution of nNOS was studied in fixed guinea-pig liver by light microscopic immunohistochemistry. Confocal analysis was used to determine its colocalization with neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP). nNOS-immunoreactive (nNOS-IR) nerves were observed in relation to hilar and interlobar vessels and in Glisson's capsule. A few nNOS-IR ganglia were observed in the extrahepatic bile duct and close to the interlobar portal triads. In addition, nNOS-IR fibers were located in the interlobular portal triads and pervading the parenchyma. Moreover, nNOS-IR nerves were demonstrated for the first time in the larger central veins and in the hepatic vein. nNOS-NPY and nNOS-CGRP colocalizations were detected in the fibromuscular layer of the bile duct and periductal plexus, respectively. These results support the phylogenetic conservation of the nNOS-IR hepatic innervation and its possible contribution to the regulation of hepatic blood flow and certain hepatic functions.

  1. Nitric oxide and vasoactive intestinal peptide as co-transmitters of airway smooth-muscle relaxation: analysis in neuronal nitric oxide synthase knockout mice.

    Science.gov (United States)

    Hasaneen, Nadia A; Foda, Hussein D; Said, Sami I

    2003-09-01

    Both vasoactive intestinal peptide (VIP) and nitric oxide (NO) relax airway smooth muscle and are potential co-transmitters of neurogenic airway relaxation. The availability of neuronal NO synthase (nNOS) knockout mice (nNOS-/-) provides a unique opportunity for evaluating NO. To evaluate the relative importance of NO, especially that generated by nNOS, and VIP as transmitters of the inhibitory nonadrenergic, noncholinergic (NANC) system. In this study, we compared the neurogenic (tetrodotoxin-sensitive) NANC relaxation of tracheal segments from nNOS-/- mice and control wild-type mice (nNOS(+/+)), induced by electrical field stimulation (EFS). We also examined the tracheal contractile response to methacholine and its relaxant response to VIP. EFS (at 60 V for 2 ms, at 10, 15, or 20 Hz) dose-dependently reduced tracheal tension, and the relaxations were consistently smaller (approximately 40%) in trachea from nNOS-/- mice than from control wild-type mice (p 0.05). Our data suggest that, in mouse trachea, NO is probably responsible for mediating a large (approximately 60%) component of neurogenic NANC relaxation, and a similar (approximately 50%) component of the relaxant effect of VIP. The results imply that NO contributes significantly to neurogenic relaxation of mouse airway smooth muscle, whether due to neurogenic stimulation or to the neuropeptide VIP.

  2. Hassallidins, antifungal glycolipopeptides, are widespread among cyanobacteria and are the end-product of a nonribosomal pathway.

    Science.gov (United States)

    Vestola, Johanna; Shishido, Tania K; Jokela, Jouni; Fewer, David P; Aitio, Olli; Permi, Perttu; Wahlsten, Matti; Wang, Hao; Rouhiainen, Leo; Sivonen, Kaarina

    2014-05-06

    Cyanobacteria produce a wide variety of cyclic peptides, including the widespread hepatotoxins microcystins and nodularins. Another class of peptides, cyclic glycosylated lipopeptides called hassallidins, show antifungal activity. Previously, two hassallidins (A and B) were reported from an epilithic cyanobacterium Hassallia sp. and found to be active against opportunistic human pathogenic fungi. Bioinformatic analysis of the Anabaena sp. 90 genome identified a 59-kb cryptic inactive nonribosomal peptide synthetase gene cluster proposed to be responsible for hassallidin biosynthesis. Here we describe the hassallidin biosynthetic pathway from Anabaena sp. SYKE748A, as well as the large chemical variation and common occurrence of hassallidins in filamentous cyanobacteria. Analysis demonstrated that 20 strains of the genus Anabaena carry hassallidin synthetase genes and produce a multitude of hassallidin variants that exhibit activity against Candida albicans. The compounds discovered here were distinct from previously reported hassallidins A and B. The IC50 of hassallidin D was 0.29-1.0 µM against Candida strains. A large variation in amino acids, sugars, their degree of acetylation, and fatty acid side chain length was detected. In addition, hassallidins were detected in other cyanobacteria including Aphanizomenon, Cylindrospermopsis raciborskii, Nostoc, and Tolypothrix. These compounds may protect some of the most important bloom-forming and globally distributed cyanobacteria against attacks by parasitic fungi.

  3. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

    Directory of Open Access Journals (Sweden)

    Roslyn D Noar

    Full Text Available Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that

  4. C-S bond cleavage by a polyketide synthase domain.

    Science.gov (United States)

    Ma, Ming; Lohman, Jeremy R; Liu, Tao; Shen, Ben

    2015-08-18

    Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.

  5. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

    Science.gov (United States)

    Noar, Roslyn D; Daub, Margaret E

    2016-01-01

    Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode

  6. Increased expression and local accumulation of the Prion Protein, Alzheimer Aβ peptides, superoxide dismutase 1, and Nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes

    Directory of Open Access Journals (Sweden)

    Bitel Claudine L

    2010-09-01

    Full Text Available Abstract Background Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP, and superoxide dismutase 1 (SOD1, as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies. Results Diabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2 in diabetic muscle. In contrast, we detected little change in Alzheimer Aβ precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aβ peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aβ cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aβ, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration. Conclusions The present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study

  7. The biosynthesis of Caryophyllaceae-like cyclic peptides in Saponaria vaccaria L. from DNA-encoded precursors.

    Science.gov (United States)

    Condie, Janet A; Nowak, Goska; Reed, Darwin W; Balsevich, J John; Reaney, Martin J T; Arnison, Paul G; Covello, Patrick S

    2011-08-01

    Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non-ribosomal peptide synthases or ribosome-dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5-9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome-dependent biosynthesis of Caryophyllaceae-like CPs in the Caryophyllaceae and Rutaceae.

  8. Pseudouridine synthases.

    Science.gov (United States)

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  9. Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

    DEFF Research Database (Denmark)

    Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus;

    2016-01-01

    .g.~antibiotics. There is thus an interest in predicting the compound synthesized by an NRPS from its primary structure (amino acid sequence) alone, as this would enable an in silico search of whole genomes for NRPS enzymes capable of synthesizing potentially useful compounds. Results: NRPS synthesis happens in a conveyor belt...

  10. Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Cairns, Timothy; Stack, Deirdre

    2011-01-01

    that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P corticosteroid model...... of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P

  11. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Siewers, Verena; Chen, Xiao; Huang, Le

    2009-01-01

    -(l-α-aminoadipyl)–l-cysteinyl–d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases...... production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5′ end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 °C. When ACVS and PPTase...... encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast....

  12. Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation

    DEFF Research Database (Denmark)

    Ramachandran, Roshni; Bhatt, Deepak Kumar; Ploug, Kenneth Beri

    2014-01-01

    -related peptide (CGRP) systems on the GTN-induced neuronal activation in this model. MATERIALS AND METHODS: The femoral vein was catheterised in rats and GTN was infused (4 µg/kg/min, for 20 minutes, intravenously). Immunohistochemistry was performed to analyse Fos, nNOS and CGRP and Western blot for measuring n...

  13. The mycosubtilin synthetase of Bacillus subtilis ATCC6633 : A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

    NARCIS (Netherlands)

    Duitman, EH; Hamoen, LW; Rembold, M; Venema, G; Seitz, H; Saenger, W; Bernhard, F; Reinhardt, R; Schmidt, M; Ullrich, C; Stein, T; Leenders, F; Vater, J

    1999-01-01

    Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a p-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Cl

  14. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  15. Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin

    Science.gov (United States)

    Palomo, Sara; González, Ignacio; de la Cruz, Mercedes; Martín, Jesús; Tormo, José Rubén; Anderson, Matthew; Hill, Russell T.; Vicente, Francisca; Reyes, Fernando; Genilloud, Olga

    2013-01-01

    Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs. PMID:23538871

  16. Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin

    Directory of Open Access Journals (Sweden)

    Olga Genilloud

    2013-03-01

    Full Text Available Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS and polyketide synthases (PKS. A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA, Acinetobacter baumannii and Candida albicans. Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs.

  17. Antimicrobial Peptides from Marine Proteobacteria

    Directory of Open Access Journals (Sweden)

    Yannick Fleury

    2013-09-01

    Full Text Available After years of inadequate use and the emergence of multidrug resistant (MDR strains, the efficiency of “classical” antibiotics has decreased significantly. New drugs to fight MDR strains are urgently needed. Bacteria hold much promise as a source of unusual bioactive metabolites. However, the potential of marine bacteria, except for Actinomycetes and Cyanobacteria, has been largely underexplored. In the past two decades, the structures of several antimicrobial compounds have been elucidated in marine Proteobacteria. Of these compounds, polyketides (PKs, synthesised by condensation of malonyl-coenzyme A and/or acetyl-coenzyme A, and non-ribosomal peptides (NRPs, obtained through the linkage of (unusual amino acids, have recently generated particular interest. NRPs are good examples of naturally modified peptides. Here, we review and compile the data on the antimicrobial peptides isolated from marine Proteobacteria, especially NRPs.

  18. Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels

    Science.gov (United States)

    Lücking, Genia; Frenzel, Elrike; Rütschle, Andrea; Marxen, Sandra; Stark, Timo D.; Hofmann, Thomas; Scherer, Siegfried; Ehling-Schulz, Monika

    2015-01-01

    The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY. PMID:26528255

  19. Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels.

    Science.gov (United States)

    Lücking, Genia; Frenzel, Elrike; Rütschle, Andrea; Marxen, Sandra; Stark, Timo D; Hofmann, Thomas; Scherer, Siegfried; Ehling-Schulz, Monika

    2015-01-01

    The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

  20. Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels

    Directory of Open Access Journals (Sweden)

    Genia eLücking

    2015-10-01

    Full Text Available The emetic toxin cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like mega-plasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the nonribosomal peptide synthetase (NRPS, gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling cereulide synthesis on a transcriptional Level, while the ABC transporter CesCD was found to be essential for post-translational control of cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

  1. First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

    Directory of Open Access Journals (Sweden)

    Patrick C Y Woo

    Full Text Available BACKGROUND: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes. METHODOLOGY/PRINCIPAL FINDINGS: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05. There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05. CONCLUSIONS/SIGNIFICANCE: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid

  2. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  3. Biochemistry: Acetohydroxyacid Synthase

    Directory of Open Access Journals (Sweden)

    Pham Ngoc Chien

    2010-02-01

    Full Text Available Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS is a thiamin-and FAD-dependent enzyme which catalyses the first common step in the biosynthesis of the branched-chain amino acids (BCAA isoleucine, leucine and valine. The enzyme is inhibited by several commercial herbicides and has been studied over the last 20 to 30 years. A short introductory note about acetohydroxyacid synthase has been provided.

  4. Diversity of Nonribosomal Peptide Synthetase Genes in the AnticancerProducing Actinomycetes Isolated from Marine Sediment in Indonesia

    OpenAIRE

    Camelia Herdini; Shinta Hartanto; Sofia Mubarika; Bambang Hariwiyanto; Nastiti Wijayanti; Akira Hosoyama; Atsushi Yamazoe; Hideaki Nojiri; Jaka Widada

    2016-01-01

    Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. ...

  5. The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus (“Penicillium islandicum”)

    DEFF Research Database (Denmark)

    Schafhauser, Thomas; Kirchner, Norbert; Kulik, Andreas

    2016-01-01

    Talaromyces islandicus (“Penicillium islandicum”) is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains...

  6. Characterization of native 40 S particles from Krebs II mouse ascites tumor cells: resolution, nomenclature and molecular weights of the nonribosomal proteins

    DEFF Research Database (Denmark)

    Reichert, G; Issinger, O G

    1981-01-01

    Native 40 S particles from Krebs II mouse ascites tumor cells were isolated on a large scale. A nonribosomal protein moiety of about 30 proteins could be removed from the ribosomal particles by treatment with 250 mM KCl. These proteins were analysed by two-dimensional polyacrylamide gel electroph......Native 40 S particles from Krebs II mouse ascites tumor cells were isolated on a large scale. A nonribosomal protein moiety of about 30 proteins could be removed from the ribosomal particles by treatment with 250 mM KCl. These proteins were analysed by two-dimensional polyacrylamide gel...

  7. Surveying the potential of secreted antimicrobial peptides to enhance plant disease resistance.

    Directory of Open Access Journals (Sweden)

    Susan eBreen

    2015-10-01

    Full Text Available Antimicrobial peptides (AMPs are natural products found across diverse taxa as part of the innate immune system against pathogen attacks. Some AMPs are synthesised through the canonical gene expression machinery and are called ribosomal AMPs. Other AMPs are assembled by modular enzymes generating nonribosomal AMPs and harbour unusual structural diversity. Plants synthesise an array of AMPs, yet are still subject to many pathogen invasions. Crop breeding programs struggle to release new cultivars in which complete disease resistance is achieved, and usually such resistance becomes quickly overcome by the targeted pathogens which have a shorter generation time. AMPs could offer a solution by exploring not only plant-derived AMPs, related or unrelated to the crop of interest, but also non-plant AMPs produced by bacteria, fungi, oomycetes or animals. This review highlights some promising candidates within the plant kingdom and elsewhere, and offers some perspectives on how to identify and validate their bioactivities. Technological advances, particularly in mass spectrometry (MS and nuclear magnetic resonance (NMR, have been instrumental in identifying and elucidating the structure of novel AMPs, especially nonribosomal peptides which cannot be identified through genomics approaches. The majority of non-plant AMPs showing potential for plant disease immunity are often tested using in vitro assays. The greatest challenge remains the functional validation of candidate AMPs in plants through transgenic experiments, particularly introducing nonribosomal AMPs into crops.

  8. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    Science.gov (United States)

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  9. Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells

    DEFF Research Database (Denmark)

    Schuck, J; Reichert, G; Issinger, O G

    1981-01-01

    Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products...

  10. Characterization of native 40 S particles from Krebs II mouse ascites tumor cells: resolution, nomenclature and molecular weights of the nonribosomal proteins

    DEFF Research Database (Denmark)

    Reichert, G; Issinger, O G

    1981-01-01

    Native 40 S particles from Krebs II mouse ascites tumor cells were isolated on a large scale. A nonribosomal protein moiety of about 30 proteins could be removed from the ribosomal particles by treatment with 250 mM KCl. These proteins were analysed by two-dimensional polyacrylamide gel electroph...

  11. Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

    National Research Council Canada - National Science Library

    Glenn Turner; Jonathan Gershenzon; Erik E. Nielson; John E. Froehlich; Rodney Croteau

    1999-01-01

    ...)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide...

  12. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  13. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  14. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...... in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...

  15. Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design

    Science.gov (United States)

    Cai, Xiaofeng; Nowak, Sarah; Wesche, Frank; Bischoff, Iris; Kaiser, Marcel; Fürst, Robert; Bode, Helge. B.

    2017-04-01

    The production of natural product compound libraries has been observed in nature for different organisms such as bacteria, fungi and plants; however, little is known about the mechanisms generating such chemically diverse libraries. Here we report mechanisms leading to the biosynthesis of the chemically diverse rhabdopeptide/xenortide peptides (RXPs). They are exclusively present in entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes delivering them to insect prey, which is killed and utilized for nutrition by both nematodes and bacteria. Chemical diversity of the biologically active RXPs results from a combination of iterative and flexible use of monomodular nonribosomal peptide synthetases including substrate promiscuity, enzyme cross-talk and enzyme stoichiometry as shown by in vivo and in vitro experiments. Together, this highlights several of nature's methods for diversification, or evolution, of natural products and sheds light on the biosynthesis of the bioactive RXPs.

  16. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  17. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  18. Influence of C-Peptide on Glucose Utilisation

    Directory of Open Access Journals (Sweden)

    B. Wilhelm

    2008-01-01

    Full Text Available During the recent years, multiple studies demonstrated that C-peptide is not an inert peptide, but exerts important physiological effects. C-peptide binds to cell membranes, stimulates the Na,K-ATPase and the endothelial nitric oxide (NO synthase. Moreover, there is evidence that C-peptide decreases glomerular hyperfiltration and increases glucose utilisation. Nevertheless, there is still limited knowledge concerning mechanisms leading to an increased glucose utilisation either in rats or in humans. The aim of this paper is to give an overview over the published studies regarding C-peptide and glucose metabolism from in vitro studies to longer lasting studies in humans.

  19. Prenyldiphosphate synthases and gibberellin biosynthesis

    NARCIS (Netherlands)

    van Schie, C.C.N.; Haring, M.A.; Schuurink, R.C.; Bach, T.J.; Rohmer, M.

    2013-01-01

    Gibberellins are derived from the diterpene precursor geranylgeranyl diphophosphate (GGPP). GGPP is converted to ent-kaurene, which contains the basic structure of gibberellins, in the plastids by the combined actions of copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Generally, ge

  20. Evolution and function of phytochelatin synthases.

    Science.gov (United States)

    Clemens, Stephan

    2006-02-01

    Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

  1. Sucrose Synthase Expression during Cold Acclimation in Wheat 1

    Science.gov (United States)

    Crespi, Martin D.; Zabaleta, Eduardo J.; Pontis, Horacio G.; Salerno, Graciela L.

    1991-01-01

    When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)+ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress. ImagesFigure 1Figure 2Figure 3 PMID:16668270

  2. Two novel cyclic peptides are key components of the antimicrobial activity of the Greenlandic isolate Pseudomonas sp. In5

    DEFF Research Database (Denmark)

    Hennessy, Rosanna Catherine; Phippen, Christopher; Nielsen, Kristian F.

    Pseudomonas sp. are a rich source of secondary metabolites including bioactive non-ribosomal peptides (NRPs) and polyketides. NRPs are synthesised in large assembly lines by multi-domain modular enzymes known as NRP-synthetases (NRPS). Nunamycin and nunapeptin are two cyclic NRPs synthesised...... by the Greenlandic isolate Pseudomonas sp. In5. Nunamycin shows antifungal activity against the basidiomycete Rhizoctonia solani whereas the only partially structure elucidated nunapeptin appears most active against the ascomycete Fusarium graminearum and the oomycete Pythium aphanidermatum. Originally isolated from...

  3. Bioactive Peptides

    Directory of Open Access Journals (Sweden)

    Eric Banan-Mwine Daliri

    2017-04-01

    Full Text Available The increased consumer awareness of the health promoting effects of functional foods and nutraceuticals is the driving force of the functional food and nutraceutical market. Bioactive peptides are known for their high tissue affinity, specificity and efficiency in promoting health. For this reason, the search for food-derived bioactive peptides has increased exponentially. Over the years, many potential bioactive peptides from food have been documented; yet, obstacles such as the need to establish optimal conditions for industrial scale production and the absence of well-designed clinical trials to provide robust evidence for proving health claims continue to exist. Other important factors such as the possibility of allergenicity, cytotoxicity and the stability of the peptides during gastrointestinal digestion would need to be addressed. This review discusses our current knowledge on the health effects of food-derived bioactive peptides, their processing methods and challenges in their development.

  4. Bioactive Peptides.

    Science.gov (United States)

    Daliri, Eric Banan-Mwine; Oh, Deog H; Lee, Byong H

    2017-04-26

    The increased consumer awareness of the health promoting effects of functional foods and nutraceuticals is the driving force of the functional food and nutraceutical market. Bioactive peptides are known for their high tissue affinity, specificity and efficiency in promoting health. For this reason, the search for food-derived bioactive peptides has increased exponentially. Over the years, many potential bioactive peptides from food have been documented; yet, obstacles such as the need to establish optimal conditions for industrial scale production and the absence of well-designed clinical trials to provide robust evidence for proving health claims continue to exist. Other important factors such as the possibility of allergenicity, cytotoxicity and the stability of the peptides during gastrointestinal digestion would need to be addressed. This review discusses our current knowledge on the health effects of food-derived bioactive peptides, their processing methods and challenges in their development.

  5. Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani

    DEFF Research Database (Denmark)

    Romans-Fuertes, Patricia; Sondergaard, Teis Esben; Sandmann, Manuela Ilse Helga

    2016-01-01

    Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate kno...... and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread....

  6. Perspectives and Insights into the Competition for Aminoacyl-tRNAs between the Translational Machinery and for tRNA Dependent Non-Ribosomal Peptide Bond Formation

    Directory of Open Access Journals (Sweden)

    Angela W. S. Fung

    2015-12-01

    Full Text Available Aminoacyl-tRNA protein transferases catalyze the transfer of amino acids from aminoacyl-tRNAs to polypeptide substrates. Different forms of these enzymes are found in the different kingdoms of life and have been identified to be central to a wide variety of cellular processes. L/F-transferase is the sole member of this class of enzyme found in Escherichia coli and catalyzes the transfer of leucine to the N-termini of proteins which result in the targeted degradation of the modified protein. Recent investigations on the tRNA specificity of L/F-transferase have revealed the unique recognition nucleotides for a preferred Leu-tRNALeu isoacceptor substrate. In addition to discussing this tRNA selectivity by L/F-transferase, we present and discuss a hypothesis and its implications regarding the apparent competition for this aminoacyl-tRNA between L/F-transferase and the translational machinery. Our discussion reveals a hypothetical involvement of the bacterial stringent response that occurs upon amino acid limitation as a potential cellular event that may reduce this competition and provide the opportunity for L/F-transferase to readily increase its access to the pool of aminoacylated tRNA substrates.

  7. Elucidation and modeling of the in-vivo kinetics of enzymes and membrane transporters associated with β-lactam and non-ribosomal peptide production in Penicillium chrysogenum

    NARCIS (Netherlands)

    Deshmukh, A.T.

    2013-01-01

    Even 80 years after the discovery of penicillin, it still holds 16% of total antibiotics market. This makes it crucial, from an economical point of view, to improve our understanding of the production organism Penicillium chrysogenum to maximize the penicillin production, as its theoretical yields a

  8. The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus (“Penicillium islandicum”)

    DEFF Research Database (Denmark)

    Schafhauser, Thomas; Kirchner, Norbert; Kulik, Andreas;

    2016-01-01

    Talaromyces islandicus (“Penicillium islandicum”) is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains ...

  9. Pseudouridines and pseudouridine synthases of the ribosome.

    Science.gov (United States)

    Ofengand, J; Malhotra, A; Remme, J; Gutgsell, N S; Del Campo, M; Jean-Charles, S; Peil, L; Kaya, Y

    2001-01-01

    psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes

  10. Tagging polyketides/non-ribosomal peptides with a clickable functionality and applications

    Directory of Open Access Journals (Sweden)

    Xuejun eZhu

    2015-02-01

    Full Text Available Bioorthogonal chemistry has recently emerged to be one of the most powerful tools in drug discovery and chemical biology. The exploration of it has successfully advanced the field of natural product research. In this Perspective, we survey current strategies for the installation of chemical handles into the molecular scaffolds of several major classes of natural products, including polyketides, non-ribosomal peptides, and their hybrids. By tagging these natural products with chemical handles and coupling them with subsequent bioorthogonal reactions, researchers have visualized and studied the mode of action of natural products, as well as synthesized derivatives with better pharmaceutical properties. We conclude this Perspective by considering two questions: Is there a general way to synthesize tagged polyketides/non-ribosomal peptides? Does natural product labeling have a broader impact in the field of natural product research beyond current known applications?

  11. Tagging polyketides/non-ribosomal peptides with a clickable functionality and applications

    Science.gov (United States)

    Zhu, Xuejun; Zhang, Wenjun

    2015-02-01

    Bioorthogonal chemistry has recently emerged to be one of the most powerful tools in drug discovery and chemical biology. The exploration of it has successfully advanced the field of natural product research. In this Perspective, we survey current strategies for the installation of chemical handles into the molecular scaffolds of several major classes of natural products, including polyketides, non-ribosomal peptides, and their hybrids. By tagging these natural products with chemical handles and coupling them with subsequent bioorthogonal reactions, researchers have visualized and studied the mode of action of natural products, as well as synthesized derivatives with better pharmaceutical properties. We conclude this Perspective by considering two questions: Is there a general way to synthesize tagged polyketides/non-ribosomal peptides? Does natural product labeling have a broader impact in the field of natural product research beyond current known applications?

  12. Peptide identification

    Science.gov (United States)

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  13. Endogenous cross-talk of fungal metabolites.

    Directory of Open Access Journals (Sweden)

    Kevin J Sheridan

    2015-01-01

    Full Text Available Non-ribosomal peptide synthesis in fungi requires a ready supply of proteogenic and non-proteogenic amino acids which are subsequently incorporated into the nascent non-ribosomal peptide via a thiotemplate mechanism catalysed by non-ribosomal peptide synthetases. Substrate amino acids can be modified prior to or during incorporation into the non-ribosomal peptide, or following incorporation into an early stage amino acid-containing biosynthetic intermediate. These post-incorporation modifications involve a range of additional enzymatic activities including but not exclusively, monooxygenases, methyltransferases, epimerases, oxidoreductases and glutathione transferases which are essential to effect biosynthesis of the final non-ribosomal peptide. Likewise, polyketide biosynthesis is directly by polyketide synthase megaenzymes and cluster-encoded ancilliary decorating enzymes. Additionally, a suite of additional primary metabolites, for example: CoA, acetyl CoA, S-adenosylmethionine, glutathione, NADPH, malonyl CoA and molecular oxygen, amongst others are required for non-ribosomal peptide and polyketide synthesis. Clearly these processes must involve exquisite orchestration to facilitate the simultaneous biosynthesis of different types of non-ribosomal peptides, polyketides, and related metabolites requiring identical or similar biosynthetic precursors or co-factors. Moreover, the near identical structures of many natural products within a given family (e.g., ergot alkaloids, along with localization to similar regions within fungi (e.g., conidia suggests that cross-talk may exist, in terms of biosynthesis and functionality. Finally, we speculate if certain biosynthetic steps involved in non-ribosomal peptide and polyketide synthesis play a role in cellular protection or environmental adaptation, and wonder if these enzymatic reactions are of equivalent importance to the actual biosynthesis of the final metabolite.

  14. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  15. Biphenyl synthase, a novel type III polyketide synthase.

    Science.gov (United States)

    Liu, B; Raeth, T; Beuerle, T; Beerhues, L

    2007-05-01

    Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

  16. Genetics Home Reference: GM3 synthase deficiency

    Science.gov (United States)

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions GM3 synthase deficiency GM3 synthase ...

  17. Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

    Science.gov (United States)

    Herbst, Dominik A; Jakob, Roman P; Zähringer, Franziska; Maier, Timm

    2016-03-24

    Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

  18. Sphingomyelin synthase SMS2 displays dual activity as ceramide phosphoethanolamine synthase[S

    Science.gov (United States)

    Ternes, Philipp; Brouwers, Jos F. H. M.; van den Dikkenberg, Joep; Holthuis, Joost C. M.

    2009-01-01

    Sphingolipids are vital components of eukaryotic membranes involved in the regulation of cell growth, death, intracellular trafficking, and the barrier function of the plasma membrane (PM). While sphingomyelin (SM) is the major sphingolipid in mammals, previous studies indicate that mammalian cells also produce the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or the enzyme(s) responsible for CPE biosynthesis. SM production is mediated by the SM synthases SMS1 in the Golgi and SMS2 at the PM, while a closely related enzyme, SMSr, has an unknown biochemical function. We now demonstrate that SMS family members display striking differences in substrate specificity, with SMS1 and SMSr being monofunctional enzymes with SM and CPE synthase activity, respectively, and SMS2 acting as a bifunctional enzyme with both SM and CPE synthase activity. In agreement with the PM residency of SMS2, we show that both SM and CPE synthase activities are enhanced at the surface of SMS2-overexpressing HeLa cells. Our findings reveal an unexpected diversity in substrate specificity among SMS family members that should enable the design of specific inhibitors to target the biological role of each enzyme individually. PMID:19454763

  19. Nunamycin and Nunapeptin: Two novel cyclic peptides are key components of the antimicrobial activity of the Greenlandic isolate Pseudomonas fluorescens In5

    DEFF Research Database (Denmark)

    Hennessy, Rosanna Catherine; Phippen, Christopher; Nielsen, Kristian F.

    Pseudomonas spp. are a rich source of secondary metabolites including bioactive non-ribosomal peptides (NRPs) and polyketides. NRPs are synthesised in large assembly lines by multi-domain modular enzymes known as NRP-synthetases (NRPS). Nunamycin and nunapeptin are two cyclic NRPs synthesised...... by the Greenlandic isolate P. fluorescens In5. Nunamycin shows antifungal activity against the basidiomycete Rhizoctonia solani whereas the only partially structure elucidated nunapeptin appears most active against the ascomycete Fusarium graminearum and the oomycete Pythium aphanidermatum. Originally isolated from...

  20. C-Peptide Test

    Science.gov (United States)

    ... AACC products and services. Advertising & Sponsorship: Policy | Opportunities C-peptide Share this page: Was this page helpful? Also known as: Insulin C-peptide; Connecting Peptide Insulin; Proinsulin C-peptide Formal ...

  1. Identification of a hybrid PKS-NRPS required for the biosynthesis of NG-391 in Metarhizium anisopliae var. anisopliae

    Science.gov (United States)

    A 19,818 kb genomic region harboring six predicted ORFs was identified in M. anisopliae ARSEF 2575. ORF4, putatively encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) was targeted using Agrobacterium-mediated gene knockout. Homologous recombinants failed to produce det...

  2. Identification of a hybrid PKS-NRPS required for the biosynthesis of NG-391 and NG-393 metabolites in Metarhizium anisopliae

    Science.gov (United States)

    A 19,818 kb genomic region harboring six predicted ORFs was identified in M. anisopliae ARSEF 2575. The ORF4 CDS, putatively encoding a hybrid polyketide synthase/nonribosomal peptide synthetase (PKS-NRPS) was targeted using Agrobacterium-mediated gene knockout. Homologous, but not heterolog...

  3. Ability of secondary metabolites from trichoderma virens to mediate communication during mutualistic or pathogenic interactions

    Science.gov (United States)

    A bioinformatic study was conducted to identify the putative genes in the biocontrol agent Trichoderma virens that encode for non-ribosomal peptide synthetases (NRPS). Gene expression analysis of 22 putative NRPSs and 4 NRPS/PKS (polyketide synthase) hybrid enzymes was conducted in the presence and...

  4. Type II thioesterase from Streptomyces coelicolor A3(2)

    NARCIS (Netherlands)

    Kotowska, Magdalena; Pawlik, Krzysztof; Butler, Andrew R.; Cundliffe, Eric; Takano, Eriko; Kuczek, Katarzyna

    2002-01-01

    Type I polyketide synthases (PKSs) are complexes of large, multimodular enzymes that catalyse biosynthesis of polyketide compounds via repetitive reaction sequences, during which each step is catalysed by a separate enzymic domain. Many type I PKSs, and also non-ribosomal peptide synthetase clusters

  5. Identification of the Biosynthetic Gene Clusters for the Lipopeptides Fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum

    DEFF Research Database (Denmark)

    Sørensen, Jens Laurids; Sondergaard, Teis Esben; Covarelli, Lorenzo;

    2014-01-01

    The closely related species Fusarium graminearum and Fusarium pseudograminearum differ in that each contains a gene cluster with a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) that is not present in the other species. To identify their products, we deleted PKS6 and NRPS7...

  6. Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

    DEFF Research Database (Denmark)

    Nielsen, Maria Lund; Petersen, Thomas Isbrandt; Petersen, Lene Maj

    2016-01-01

    Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives...

  7. Peptide arrays for screening cancer specific peptides.

    Science.gov (United States)

    Ahmed, Sahar; Mathews, Anu Stella; Byeon, Nara; Lavasanifar, Afsaneh; Kaur, Kamaljit

    2010-09-15

    In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis.

  8. Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction.

    Science.gov (United States)

    Abe, Tomoko; Hashimoto, Yoshiteru; Zhuang, Ye; Ge, Yin; Kumano, Takuto; Kobayashi, Michihiko

    2016-01-22

    We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.

  9. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  10. The majority of total nuclear-encoded non-ribosomal RNA in a human cell is 'dark matter' un-annotated RNA

    Directory of Open Access Journals (Sweden)

    Milos Patrice

    2010-12-01

    Full Text Available Abstract Background Discovery that the transcriptional output of the human genome is far more complex than predicted by the current set of protein-coding annotations and that most RNAs produced do not appear to encode proteins has transformed our understanding of genome complexity and suggests new paradigms of genome regulation. However, the fraction of all cellular RNA whose function we do not understand and the fraction of the genome that is utilized to produce that RNA remain controversial. This is not simply a bookkeeping issue because the degree to which this un-annotated transcription is present has important implications with respect to its biologic function and to the general architecture of genome regulation. For example, efforts to elucidate how non-coding RNAs (ncRNAs regulate genome function will be compromised if that class of RNAs is dismissed as simply 'transcriptional noise'. Results We show that the relative mass of RNA whose function and/or structure we do not understand (the so called 'dark matter' RNAs, as a proportion of all non-ribosomal, non-mitochondrial human RNA (mt-RNA, can be greater than that of protein-encoding transcripts. This observation is obscured in studies that focus only on polyA-selected RNA, a method that enriches for protein coding RNAs and at the same time discards the vast majority of RNA prior to analysis. We further show the presence of a large number of very long, abundantly-transcribed regions (100's of kb in intergenic space and further show that expression of these regions is associated with neoplastic transformation. These overlap some regions found previously in normal human embryonic tissues and raises an interesting hypothesis as to the function of these ncRNAs in both early development and neoplastic transformation. Conclusions We conclude that 'dark matter' RNA can constitute the majority of non-ribosomal, non-mitochondrial-RNA and a significant fraction arises from numerous very long

  11. Phytochelatins: peptides involved in heavy metal detoxification.

    Science.gov (United States)

    Pal, Rama; Rai, J P N

    2010-03-01

    Phytochelatins (PCs) are enzymatically synthesized peptides known to involve in heavy metal detoxification and accumulation, which have been measured in plants grown at high heavy metal concentrations, but few studies have examined the response of plants even at lower environmentally relevant metal concentrations. Recently, genes encoding the enzyme PC synthase have been identified in plants and other species enabling molecular biological studies to untangle the mechanisms underlying PC synthesis and its regulation. The present paper embodies review on recent advances in structure of PCs, their biosynthetic regulation, roles in heavy metal detoxification and/or accumulation, and PC synthase gene expression for better understanding of mechanism involved and to improve phytoremediation efficiency of plants for wider application.

  12. Homocysteine Editing, Thioester Chemistry, Coenzyme A, and the Origin of Coded Peptide Synthesis †.

    Science.gov (United States)

    Jakubowski, Hieronim

    2017-02-09

    Aminoacyl-tRNA synthetases (AARSs) have evolved "quality control" mechanisms which prevent tRNA aminoacylation with non-protein amino acids, such as homocysteine, homoserine, and ornithine, and thus their access to the Genetic Code. Of the ten AARSs that possess editing function, five edit homocysteine: Class I MetRS, ValRS, IleRS, LeuRS, and Class II LysRS. Studies of their editing function reveal that catalytic modules of these AARSs have a thiol-binding site that confers the ability to catalyze the aminoacylation of coenzyme A, pantetheine, and other thiols. Other AARSs also catalyze aminoacyl-thioester synthesis. Amino acid selectivity of AARSs in the aminoacyl thioesters formation reaction is relaxed, characteristic of primitive amino acid activation systems that may have originated in the Thioester World. With homocysteine and cysteine as thiol substrates, AARSs support peptide bond synthesis. Evolutionary origin of these activities is revealed by genomic comparisons, which show that AARSs are structurally related to proteins involved in coenzyme A/sulfur metabolism and non-coded peptide bond synthesis. These findings suggest that the extant AARSs descended from ancestral forms that were involved in non-coded Thioester-dependent peptide synthesis, functionally similar to the present-day non-ribosomal peptide synthetases.

  13. Homocysteine Editing, Thioester Chemistry, Coenzyme A, and the Origin of Coded Peptide Synthesis †

    Directory of Open Access Journals (Sweden)

    Hieronim Jakubowski

    2017-02-01

    Full Text Available Aminoacyl-tRNA synthetases (AARSs have evolved “quality control” mechanisms which prevent tRNA aminoacylation with non-protein amino acids, such as homocysteine, homoserine, and ornithine, and thus their access to the Genetic Code. Of the ten AARSs that possess editing function, five edit homocysteine: Class I MetRS, ValRS, IleRS, LeuRS, and Class II LysRS. Studies of their editing function reveal that catalytic modules of these AARSs have a thiol-binding site that confers the ability to catalyze the aminoacylation of coenzyme A, pantetheine, and other thiols. Other AARSs also catalyze aminoacyl-thioester synthesis. Amino acid selectivity of AARSs in the aminoacyl thioesters formation reaction is relaxed, characteristic of primitive amino acid activation systems that may have originated in the Thioester World. With homocysteine and cysteine as thiol substrates, AARSs support peptide bond synthesis. Evolutionary origin of these activities is revealed by genomic comparisons, which show that AARSs are structurally related to proteins involved in coenzyme A/sulfur metabolism and non-coded peptide bond synthesis. These findings suggest that the extant AARSs descended from ancestral forms that were involved in non-coded Thioester-dependent peptide synthesis, functionally similar to the present-day non-ribosomal peptide synthetases.

  14. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  15. Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

    Science.gov (United States)

    Han, Yingying; Zhao, Wenwen; Wang, Zhicui; Zhu, Jingying; Liu, Qisong

    2014-06-01

    Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

  16. Identification and characterization of a second isogene encoding γ-terpinene synthase in Thymus caespititius.

    Science.gov (United States)

    Mendes, Marta D; Barroso, José G; Oliveira, M Margarida; Trindade, Helena

    2014-07-15

    Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high polymorphism. They consist mostly of mono- and sesquiterpene, some of them with interest for the pharmaceutical and food industries. The search for terpene synthase genes was performed in three in vitro T. caespititius genotypes. For these plants, the expression of a previously described γ-terpinene synthase gene, Tctps2, was confirmed, occurring concomitantly with a new gene encoding an enzyme with similar activity, named Thymus caespititius terpene synthase 4 (Tctps4). The two isogenes were isolated and functionally characterized in the three plant genotypes. Alignment of the two Tctps revealed a transit peptide much shorter in Tctps4 than in Tctps2 (3-4 amino acids instead of 47). The Tctps4 open reading frame is shorter than Tctps2 (1665 bp versus 1794 bp). The amino acid sequence of both γ-terpinene synthases shared an 88% pairwise identity. The fact that T. caespititius carries two isogenes for γ-terpinene synthases, suggests gene duplication along the evolutionary process, followed by mutations leading to the differentiation of both genes. These mutations didn't compromise protein activity. A high accumulation of transcripts from both genes was found in shoots of in vitro plantlets, while in roots they could not be detected. Still, γ-terpinene levels in aerial parts were reduced, probably due to fast conversion into carvacrol and thymol, the main components from T. caespititius essential oils. This study is a contribution to the identification of terpene synthase genes in Lamiaceae.

  17. Structure of Salmonella typhimurium OMP synthase in a complete substrates complex

    Science.gov (United States)

    Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A.; Almo, Steven C.

    2012-01-01

    Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, E.C. 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been co-crystallized in a complete substrate complex of E•MgPRPP•orotate, and the structure solved to 2.2 Å resolution. This structure resembles that for Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands, but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99–109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, due to movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg2+ in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase, and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substratea-ssisted stabilization of the previously documented oxocarbenium transition state structure is proposed. PMID:22531064

  18. Biosynthetic regulation of phytochelatins, heavy metal-binding peptides.

    Science.gov (United States)

    Hirata, Kazumasa; Tsuji, Naoki; Miyamoto, Kazuhisa

    2005-12-01

    Phytochelatins (PCs) are heavy metal-binding peptides that play important roles in the detoxification of toxic heavy metals and the regulation of intracellular concentrations of essential metals in eukaryotes, including higher plants, fungi, and microalgae. Recently, PC synthase genes in higher plants and fission yeast have been identified and characterized, enabling molecular biological studies to unravel the mechanisms underlying PC synthesis. Moreover, recent routine database searches have unexpectedly identified genes that are similar to plant PC synthase genes in the genomes of worms and some prokaryotes. In this review, we introduce these recent advances in our understanding of the molecular mechanisms for PC biosynthesis and functions in order to supply basic information about the unique and attractive peptides applicable to various fields.

  19. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2, wh...

  20. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen;

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2...

  1. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    OpenAIRE

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic ch...

  2. The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system.

    Science.gov (United States)

    Ladenstein, Rudolf; Fischer, Markus; Bacher, Adelbert

    2013-06-01

    The xylene ring of riboflavin (vitamin B2 ) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

  3. Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis

    Science.gov (United States)

    Benjdia, Alhosna; Guillot, Alain; Ruffié, Pauline; Leprince, Jérôme; Berteau, Olivier

    2017-07-01

    Ribosomally synthesized peptides are built out of L-amino acids, whereas D-amino acids are generally the hallmark of non-ribosomal synthetic processes. Here we show that the model bacterium Bacillus subtilis is able to produce a novel type of ribosomally synthesized and post-translationally modified peptide that contains D-amino acids, and which we propose to call epipeptides. We demonstrate that a two [4Fe-4S]-cluster radical S-adenosyl-L-methionine (SAM) enzyme converts L-amino acids into their D-counterparts by catalysing Cα-hydrogen-atom abstraction and using a critical cysteine residue as the hydrogen-atom donor. Unexpectedly, these D-amino acid residues proved to be essential for the activity of a peptide that induces the expression of LiaRS, a major component of the bacterial cell envelope stress-response system. Present in B. subtilis and in several members of the human microbiome, these epipeptides and radical SAM epimerases broaden the landscape of peptidyl structures accessible to living organisms.

  4. Antimicrobial peptides of the genus Bacillus: a new era for antibiotics.

    Science.gov (United States)

    Sumi, Chandra Datta; Yang, Byung Wook; Yeo, In-Cheol; Hahm, Young Tae

    2015-02-01

    The rapid onset of resistance reduces the efficacy of most conventional antimicrobial drugs and is a general cause of concern for human well-being. Thus, there is great demand for a continuous supply of novel antibiotics to combat this problem. Bacteria-derived antimicrobial peptides (AMPs) have long been used as food preservatives; moreover, prior to the development of conventional antibiotics, these AMPs served as an efficient source of antibiotics. Recently, peptides produced by members of the genus Bacillus were shown to have a broad spectrum of antimicrobial activity against pathogenic microbes. Bacillus-derived AMPs can be synthesized both ribosomally and nonribosomally and can be classified according to peptide biosynthesis, structure, and molecular weight. The precise mechanism of action of these AMPs is not yet clear; however, one proposed mechanism is that these AMPs kill bacteria by forming channels in and (or) disrupting the bacterial cell wall. Bacillus-derived AMPs have potential in the pharmaceutical industry, as well as the food and agricultural sectors. Here, we focus on Bacillus-derived AMPs as a novel alternative approach to antibacterial drug development. We also provide an overview of the biosynthesis, mechanisms of action, applications, and effectiveness of different AMPs produced by members of the Bacillus genus, including several recently identified novel AMPs.

  5. Mammalian N-acetylglutamate synthase.

    Science.gov (United States)

    Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

    2004-04-01

    N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

  6. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  7. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

    Directory of Open Access Journals (Sweden)

    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  8. A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity.

    Science.gov (United States)

    Harada, Emiko; von Roepenack-Lahaye, Edda; Clemens, Stephan

    2004-12-01

    Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.

  9. The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

    Science.gov (United States)

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2015-12-01

    Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

  10. Critical aspartic acid residues in pseudouridine synthases.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Paulson, J L; Spedaliere, C J; Mueller, E G

    1999-08-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

  11. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides.

    Science.gov (United States)

    Hartwig, S; Frister, T; Alemdar, S; Li, Z; Scheper, T; Beutel, S

    2015-03-20

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L(-1) were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni(2+)-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg(2+) containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC-MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis-Menten model, kinetic parameters of KM = 1.111 μM (±0.113), vmax = 0.3245 μM min(-1) (±0.0035), kcat = 2.95 min(-1), as well as a catalytic efficiency kcat/KM = 4.43 × 10(4) M(-1)s(-1) were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  13. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  14. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis

    Science.gov (United States)

    Osberger, Thomas J.; Rogness, Donald C.; Kohrt, Jeffrey T.; Stepan, Antonia F.; White, M. Christina

    2016-09-01

    Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C-H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C-H oxidation strategy is demonstrated in its capacity for generating diversity: four ‘chiral pool’ amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C-H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C-H oxidation to one containing a linear unnatural amino acid.

  15. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  16. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  17. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  18. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

    Full Text Available This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

  19. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  20. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  1. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  2. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  3. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides

    Energy Technology Data Exchange (ETDEWEB)

    Hartwig, S.; Frister, T.; Alemdar, S.; Li, Z.; Scheper, T.; Beutel, S., E-mail: beutel@iftc.uni-hannover.de

    2015-03-20

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L{sup −1} were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni{sup 2+}-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg{sup 2+} containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of K{sub M} = 1.111 μM (±0.113), v{sub max} = 0.3245 μM min{sup −1} (±0.0035), k{sub cat} = 2.95 min{sup −1}, as well as a catalytic efficiency k{sub cat}/K{sub M} = 4.43 × 10{sup 4} M{sup −1} s{sup −1} were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. - Highlights: • Uncharacterized (+)-zizaene synthase from C. zizanoides was cloned

  4. The Structural Basis of [beta]-Peptide-Specific Cleavage by the Serine Protease Cyanophycinase

    Energy Technology Data Exchange (ETDEWEB)

    Law, Adrienne M.; Lai, Sandy W.S.; Tavares, John; Kimber, Matthew S.; (Guelph)

    2010-10-01

    Cyanophycin, or poly-L-Asp-multi-L-Arg, is a non-ribosomally synthesized peptidic polymer that is used for nitrogen storage by cyanobacteria and other select eubacteria. Upon synthesis, it self-associates to form insoluble granules, the degradation of which is uniquely catalyzed by a carboxy-terminal-specific protease, cyanophycinase. We have determined the structure of cyanophycinase from the freshwater cyanobacterium Synechocystis sp. PCC6803 at 1.5-{angstrom} resolution, showing that the structure is dimeric, with individual protomers resembling aspartyl dipeptidase. Kinetic characterization of the enzyme demonstrates that the enzyme displays Michaelis-Menten kinetics with a k{sub cat} of 16.5 s{sup -1} and a k{sub cat}/K{sub M} of 7.5 x 10{sup -6} M{sup -1} s{sup -1}. Site-directed mutagenesis experiments confirm that cyanophycinase is a serine protease and that Gln101, Asp172, Gln173, Arg178, Arg180 and Arg183, which form a conserved pocket adjacent to the catalytic Ser132, are functionally critical residues. Modeling indicates that cyanophycinase binds the {beta}-Asp-Arg dipeptide residue immediately N-terminal to the scissile bond in an extended conformation in this pocket, primarily recognizing this penultimate {beta}-Asp-Arg residue of the polymeric chain. Because binding and catalysis depend on substrate features unique to {beta}-linked aspartyl peptides, cyanophycinase is able to act within the cytosol without non-specific cleavage events disrupting essential cellular processes.

  5. The tomato terpene synthase gene family

    NARCIS (Netherlands)

    Falara, V.; Akhtar, T.A.; Nguyen, T.T.H.; Spyropoulou, E.A.; Bleeker, P.M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M.E.; Schilmiller, A.L.; Last, R.L.; Schuurink, R.C.; Pichersky, E.

    2011-01-01

    Compounds of the terpenoid class play many roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of Solanum lycopersicum (cultivated tomato) contains 40 terpene synthase (TPS) genes, including 28

  6. Cloning of parsley flavone synthase I.

    Science.gov (United States)

    Martens, S; Forkmann, G; Matern, U; Lukacin, R

    2001-09-01

    A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.

  7. Inducible nitric oxide synthase in renal transplantation

    NARCIS (Netherlands)

    Joles, JA; Vos, IH; Grone, HJ; Rabelink, TJ

    2002-01-01

    The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the induc

  8. Characterization of the Phytochelatin Synthase from the Human Parasitic Nematode Ancylostoma ceylanicum

    Science.gov (United States)

    Rigouin, Coraline; Vermeire, Jon J.; Nylin, Elyse; Williams, David L.

    2013-01-01

    Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention. PMID:23916800

  9. Exercise training upregulates nitric oxide synthases in the kidney of rats with chronic heart failure.

    Science.gov (United States)

    Ito, Daisuke; Ito, Osamu; Mori, Nobuyoshi; Cao, Pengyu; Suda, Chihiro; Muroya, Yoshikazu; Hao, Kiyotaka; Shimokawa, Hiroaki; Kohzuki, Masahiro

    2013-09-01

    There is an interaction between heart and kidney diseases, which is a condition termed cardiorenal syndrome. Exercise training has cardioprotective effects, involving upregulation of endothelial (e) nitric oxide synthase (NOS) in the cardiovascular system. However, the effects of exercise training on NOS in the kidney with heart disease are unknown. The aim of the present study was to investigate whether exercise training upregulates NOS in the kidney, left ventricle and aorta of rats with chronic heart failure (CHF). Male Sprague-Dawley rats underwent left coronary artery ligation (LCAL) to induce CHF and were randomly assigned to sedentary or treadmill exercise groups 4 weeks after LCAL. Three days after exercising for 4 weeks, urine samples were collected for 24 h and blood samples were collected following decapitation. Nitric oxide synthase activity and protein expression were examined. Significant interactions between CHF and exercise training were observed on parameters of cardiac and renal function. Exercise training improved cardiac function, decreased plasma B-type natriuretic peptide levels, decreased urinary albumin excretion and increased creatinine clearance in CHF rats. Nitric oxide synthase activity, eNOS expression and neuronal (n) NOS expression were significantly decreased in the left ventricle and kidney of CHF rats. Exercise training significantly increased NOS activity and eNOS and nNOS expression. Upregulation of NOS in the kidney and left ventricle may contribute, in part, to the renal and cardiac protective effects of exercise training in cardiorenal syndrome in CHF rats.

  10. Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.

    Science.gov (United States)

    Rigouin, Coraline; Vermeire, Jon J; Nylin, Elyse; Williams, David L

    2013-09-01

    Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependent and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.

  11. Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

    Science.gov (United States)

    Jagielska, Elżbieta; Płucienniczak, Andrzej; Dąbrowska, Magdalena; Dowierciał, Anna; Rode, Wojciech

    2014-04-09

    Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis. Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed. Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae. Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.

  12. D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea.

    Science.gov (United States)

    Riederer, B; Han, M; Keller, U

    1996-11-01

    The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4'-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of approximately 1.4 microM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

  13. Molecular, chemical and biological screening of soil actinomycete isolates in seeking bioactive peptide metabolites

    Directory of Open Access Journals (Sweden)

    Javad Hamedi

    2015-10-01

    Full Text Available Background and Objective: Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the interests of researchers. In this study, molecular, chemical and biological screening of soil actinomycetes was carried out in order to search for peptide-producing actinomycetes.Materials and Methods: 60 actinomycetes were isolated from soils of Iran. The isolates were subjected to molecular screening for detection NRPS (non-ribosomal peptide synthetases gene. Phylogenic identification of NRPS containing isolates was performed. Chemical screening of the crude extracts was performed using chlorine o-dianisidine as peptide detector reagent and bioactivity of peptide producing strains was determined by antimicrobial bioassay. High pressure liquid chromatography- mass spectrometry (HPLC-MS with UV-visible spectroscopy was performed for detection of the metabolite diversity in selected strain.Results: Amplified NRPS adenylation gene (700 bp was detected among 30 strains. Phylogenic identification of these isolates showed presence of rare actinomycetes genera among the isolates and 10 out of 30 strains were subjected to chemical screening. Nocardia sp. UTMC 751 showed antimicrobial activity against bacterial and fungal test pathogens. HPLC-MSand UV-visible spectroscopy results from the crude extract showed that this strain has probably the ability to produce new metabolites.Conclusion: By application of a combined approach, including molecular, chemical and bioactivity analysis, a promising strain of Nocardia sp. UTMC 751 was obtained. This strain had significant activity against Staphylococcus aureus and Pseudomonas aeruginosa. Strain Nocardia sp. UTMC 751 produce five unknown and most probably new metabolites with molecular weights of 274.2, 390.3, 415.3, 598.4 and 772.5. This strain had showed 99% similarity to Nocardia ignorata DSM 44496 T.

  14. Pep2Path: automated mass spectrometry-guided genome mining of peptidic natural products.

    Directory of Open Access Journals (Sweden)

    Marnix H Medema

    2014-09-01

    Full Text Available Nonribosomally and ribosomally synthesized bioactive peptides constitute a source of molecules of great biomedical importance, including antibiotics such as penicillin, immunosuppressants such as cyclosporine, and cytostatics such as bleomycin. Recently, an innovative mass-spectrometry-based strategy, peptidogenomics, has been pioneered to effectively mine microbial strains for novel peptidic metabolites. Even though mass-spectrometric peptide detection can be performed quite fast, true high-throughput natural product discovery approaches have still been limited by the inability to rapidly match the identified tandem mass spectra to the gene clusters responsible for the biosynthesis of the corresponding compounds. With Pep2Path, we introduce a software package to fully automate the peptidogenomics approach through the rapid Bayesian probabilistic matching of mass spectra to their corresponding biosynthetic gene clusters. Detailed benchmarking of the method shows that the approach is powerful enough to correctly identify gene clusters even in data sets that consist of hundreds of genomes, which also makes it possible to match compounds from unsequenced organisms to closely related biosynthetic gene clusters in other genomes. Applying Pep2Path to a data set of compounds without known biosynthesis routes, we were able to identify candidate gene clusters for the biosynthesis of five important compounds. Notably, one of these clusters was detected in a genome from a different subphylum of Proteobacteria than that in which the molecule had first been identified. All in all, our approach paves the way towards high-throughput discovery of novel peptidic natural products. Pep2Path is freely available from http://pep2path.sourceforge.net/, implemented in Python, licensed under the GNU General Public License v3 and supported on MS Windows, Linux and Mac OS X.

  15. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...... endothelium is consistent with a role for NO in the control of blood flow in human skeletal muscle....

  16. Acylation of Therapeutic Peptides

    DEFF Research Database (Denmark)

    Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

    peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... stems from a synergy between the positive peptide charge and membrane-active acyl moiety, supported by its pH-dependency, whereby the effect increased with decreasing pH and concomitant charge increase. The extent of permeation enhancing effect was highly dependent on acylation chain length and position...

  17. Topical peptides as cosmeceuticals

    Directory of Open Access Journals (Sweden)

    Varadraj Vasant Pai

    2017-01-01

    Full Text Available Peptides are known to have diverse biological roles, most prominently as signaling/regulatory molecules in a broad variety of physiological processes including defense, immunity, stress, growth, homeostasis and reproduction. These aspects have been used in the field of dermatology and cosmetology to produce short, stable and synthetic peptides for extracellular matrix synthesis, pigmentation, innate immunity and inflammation. The evolution of peptides over the century, which started with the discovery of penicillin, has now extended to their usage as cosmeceuticals in recent years. Cosmeceutical peptides may act as signal modulators of the extracellular matrix component, as structural peptides, carrier peptides and neurotransmitter function modulators. Transdermal delivery of peptides can be made more effective by penetration enhancers, chemical modification or encapsulation of peptides. The advantages of using peptides as cosmeceuticals include their involvement in many physiological functions of the skin, their selectivity, their lack of immunogenicity and absence of premarket regulatory requirements for their use. However, there are disadvantages: clinical evidence for efficacy is often weak, absorption may be poor due to low lipophilicity, high molecular weight and binding to other ingredients, and prices can be quite high.

  18. Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

    Science.gov (United States)

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

  19. BIOINFORMATICS AND BIOSYNTHESIS ANALYSIS OF CELLULOSE SYNTHASE OPERON IN ZYMOMONAS MOBILIS ZM4

    Directory of Open Access Journals (Sweden)

    Sheik Abdul Kader Sheik Asraf, K. Narayanan Rajnish, and Paramasamy Gunasekaran

    2011-03-01

    Full Text Available Biosynthesis of cellulose has been reported in many species of bacteria. The genes encoding cellulose biosynthetic enzymes of Z. mobilis have not been studied so far. Preliminary sequence analysis of the Z. mobilis ZM4 genome revealed the presence of a cellulose synthase operon comprised of Open Reading Frames (ORFs ZMO01083 (bcsA, ZMO1084 (bcsB and ZMO1085 (bcsC. The first gene of the operon bcsA encodes the cellulose synthase catalytic subunit BcsA. The second gene of the operon bcsB encodes the cellulose synthase subunit B (BcsB, which shows the presence of BcsB multi-domain and is inferred to bind c-di-GMP, the regulator of cellulose biosynthesis. The third gene of the operon bcsC encodes the cellulose synthase operon C domain protein (BcsC, which belongs to super family of teratrico peptide repeat (TPR that are believed to mediate protein – protein interactions for the formation of cellulose. Multiple sequence alignment of the deduced amino acid sequences of BcsA and BcsC with other closely related homologs showed the presence of PVDPYE, HAKAGNLN, DCD motif and TPR motif, the characteristic motifs of bacterial cellulose synthases. Analysis of the nucleotide sequence of the ORF ZMO1085 and neighboring ORFs namely ZMO1083 and ZMO1084 indicated that all the ORFs are translationally linked and form an operon. Transcript analysis using Real-time PCR indicated the expression of the genes involved in cellulose synthase operon in Zymomonas mobilis ZM4. Z. mobilis colonies grown on RM-glucose containing Congo red displayed a characteristic bright red-brown colour. Z. mobilis colonies grown on RM-glucose medium supplemented with Calcoflour exhibited fluorescence. The arrangement of Calcofluor stained microfibrils can be seen in fluorescence microscopy which is an indicative for cellulose biosynthesis. AFM micrograph of the extracellular matrix of Z. mobilis shows a relatively dense matrix with bacterial cell residues. The presence of cellulose was

  20. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins.

    Science.gov (United States)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph; Krogsgård Nielsen, Christina; Meyer, Rikke Louise; Slavetinsky, Christoph; Peschel, Andreas; Ingmer, Hanne

    2017-09-05

    Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available

  1. A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

    Science.gov (United States)

    Degola, Francesca; De Benedictis, Maria; Petraglia, Alessandro; Massimi, Alberto; Fattorini, Laura; Sorbo, Sergio; Basile, Adriana; Sanità di Toppi, Luigi

    2014-11-01

    Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

  2. Cellulose Synthases and Synthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Anne Endler; Staffan Persson

    2011-01-01

    Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.

  3. Caffeine synthase and related methyltransferases in plants.

    Science.gov (United States)

    Misako, Kato; Kouichi, Mizuno

    2004-05-01

    Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in high concentrations in tea and coffee and it is also found in a number of beverages such as coca cola. It is necessary to elucidate the caffeine biosynthetic pathway and to clone the genes related to the production of caffeine not only to determine the metabolism of the purine alkaloid but also to control the content of caffeine in tea and coffee. The available data support the operation of a xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway as the major route to caffeine. Since the caffeine biosynthetic pathway contains three S-adenosyl-L-methionine (SAM) dependent methylation steps, N-methyltransferases play important roles. This review focuses on the enzymes and genes involved in the methylation of purine ring. Caffeine synthase, the SAM-dependent methyltransferase involved in the last two steps of caffeine biosynthesis, was originally purified from young tea leaves (Camellia sinensis). The isolated cDNA, termed TCS1, consists of 1,483 base pairs and encodes a protein of 369 amino acids. Subsequently, the homologous genes that encode caffeine biosynthetic enzymes from coffee (Coffea arabica) were isolated. The recombinant proteins are classified into the three types on the basis of their substrate specificity i.e. 7-methylxanthosine synthase, theobromine synthase and caffeine synthase. The predicted amino acid sequences of caffeine biosynthetic enzymes derived from C. arabica exhibit more than 80% homology with those of the clones and but show only 40% homology with TCS1 derived from C. sinensis. In addition, they share 40% homology with the amino acid sequences of salicylic carboxyl methyltransferase, benzoic acid carboxyl methyltransferase and jasmonic acid carboxyl methyltransferase which belong to a family of motif B' methyltransferases which are novel plant methyltransferases with motif B' instead of motif B as the conserved region.

  4. Building-block selectivity of polyketide synthases.

    Science.gov (United States)

    Liou, Grace F; Khosla, Chaitan

    2003-04-01

    For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.

  5. Insulin C-peptide test

    Science.gov (United States)

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  6. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.;

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields.......Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  7. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  8. Avian host defense peptides

    NARCIS (Netherlands)

    Cuperus, Tryntsje; Coorens, M.; van Dijk, A.; Haagsman, H.P.

    2013-01-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense

  9. Bacteriocin Inducer Peptides

    Science.gov (United States)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  10. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase.

    Science.gov (United States)

    Bohlmann, J; Steele, C L; Croteau, R

    1997-08-29

    Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA

  11. APD: the Antimicrobial Peptide Database

    OpenAIRE

    Wang, Zhe; Wang, Guangshun

    2004-01-01

    An antimicrobial peptide database (APD) has been established based on an extensive literature search. It contains detailed information for 525 peptides (498 antibacterial, 155 antifungal, 28 antiviral and 18 antitumor). APD provides interactive interfaces for peptide query, prediction and design. It also provides statistical data for a select group of or all the peptides in the database. Peptide information can be searched using keywords such as peptide name, ID, length, net charge, hydrophob...

  12. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  13. KirCII- promising tool for polyketide diversification

    DEFF Research Database (Denmark)

    Musiol-Kroll, Ewa Maria; Härtner, Thomas; Kulik, Andreas

    2014-01-01

    Kirromycin is produced by Streptomyces collinus Tü 365. This compound is synthesized by a large assembly line of type I polyketide synthases and non-ribosomal peptide synthetases (PKS I/NRPS), encoded by the genes kirAI-kirAVI and kirB. The PKSs KirAI-KirAV have no acyltransferase domains integra...... introducing the non-native substrates in an in vivo context. Thus, KirCII represents a promising tool for polyketide diversification....

  14. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis.

    Science.gov (United States)

    Hyatt, David C; Croteau, Rodney

    2005-07-15

    Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.

  15. Structural Studies of a Complex Between Endothelial Nitric Oxide Synthase and Calmodulin at Physiological Calcium Concentration.

    Science.gov (United States)

    Piazza, Michael; Dieckmann, Thorsten; Guillemette, Joseph Guy

    2016-10-04

    The small acidic protein Calmodulin (CaM) serves as a Ca(2+) sensor and control element for many enzymes including nitric oxide synthase (NOS) enzymes that play major roles in key physiological and pathological processes. CaM binding causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. In this report, NMR spectroscopy was used to determine the solution structure of the endothelial NOS (eNOS) peptide in complex with CaM at the lowest Ca(2+) concentration (225 nM) required for CaM to bind to eNOS and corresponds to a physiological elevated Ca2+ level found in mammalian cells. Under these conditions, the CaM-eNOS complex has a Ca(2+)-replete C-terminal lobe bound the eNOS peptide and a Ca(2+) free N-terminal lobe loosely associated to the eNOS peptide. With increasing Ca(2+) concentration, the binding of Ca(2+) by the N-lobe of CaM results in a stronger interaction with the C-terminal region of the eNOS peptide and increased α-helical structure of the peptide that may be part of the mechanism resulting in electron transfer from the FMN to the heme in the oxygenase domain of the enzyme. SPR studies performed under the same conditions show Ca(2+) concentration dependent binding kinetics were consistent with the NMR structural results. This investigation shows that structural studies performed under more physiological relevant conditions provide information on subtle changes in structure that may not be apparent when experiments are performed in excess Ca(2+) concentrations.

  16. Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants.

    Science.gov (United States)

    Gray, Dennis W; Breneman, Steven R; Topper, Lauren A; Sharkey, Thomas D

    2011-06-10

    2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.

  17. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  18. Descriptors for antimicrobial peptides

    DEFF Research Database (Denmark)

    Jenssen, Håvard

    2011-01-01

    Introduction: A frightening increase in the number of isolated multidrug resistant bacterial strains linked to the decline in novel antimicrobial drugs entering the market is a great cause for concern. Cationic antimicrobial peptides (AMPs) have lately been introduced as a potential new class...... examples of different peptide QSAR studies, this review highlights some of the missing links and illuminates some of the questions that would be interesting to challenge in a more systematic fashion. Expert opinion: Computer-aided peptide QSAR using molecular descriptors may provide the necessary edge...

  19. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  20. Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity.

    Science.gov (United States)

    Li, Yujing; Dhankher, Om Parkash; Carreira, Laura; Lee, David; Chen, Alice; Schroeder, Julian I; Balish, Rebecca S; Meagher, Richard B

    2004-12-01

    Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.

  1. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives.

  2. The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

    Science.gov (United States)

    Petraglia, Alessandro; De Benedictis, Maria; Degola, Francesca; Pastore, Giovanni; Calcagno, Margherita; Ruotolo, Roberta; Mengoni, Alessio; Sanità di Toppi, Luigi

    2014-03-01

    Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 μM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

  3. (3R)-Linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound induction.

    Science.gov (United States)

    Jia, J W; Crock, J; Lu, S; Croteau, R; Chen, X Y

    1999-12-01

    Artemisia annua is an annual herb used in traditional Chinese medicine. A cDNA library was constructed from leaves of A. annua seedlings and target sequences were amplified by PCR using degenerate primers derived from a consensus sequence of angiosperm terpene synthases. Two clones, QH1 and QH5, with high sequence similarity to plant monoterpene synthases were ultimately obtained and expressed in Escherichia coli. These cDNAs encode peptides of 567 aa (65.7 kDa) and 583 aa (67.4 kDa), respectively, and display 88% identity with each other and 42% identity with Mentha spicata limonene synthase. The two recombinant enzymes yielded no detectable activity with isopentenyl diphosphate, dimethylallyl diphosphate, chrysanthemyl diphosphate, farnesyl diphosphate, (+)-copalyl diphosphate, or geranylgeranyl diphosphate, but were active with geranyl diphosphate in yielding (3R)-linalool as the sole product in the presence of divalent metal cation cofactors. QH1-linalool synthase displays a K(m) value of 64 microM for geranyl diphosphate, which is considerably higher than other known monoterpene synthases, and a K(m) value of 4.6 mM for Mg(+2). Transcripts of QH1 and QH5 could be detected by RT-PCR in the leaves and inflorescence of A. annua, but not in the stem stele or roots; transcripts of QH5 could also be detected in stem epidermis. Linalool could not be detected by GC-MS in the essential oil of A. annua, nor in acid or base hydrolysates of aqueous extracts of leaves. RT-PCR demonstrated a wound-inducible increase in QH1 and QH5 transcript abundance in both leaves and stems over a 3-day time course. Copyright 1999 Academic Press.

  4. Diversity-oriented peptide stapling

    DEFF Research Database (Denmark)

    Tran, Thu Phuong; Larsen, Christian Ørnbøl; Røndbjerg, Tobias

    2017-01-01

    as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides...

  5. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    OpenAIRE

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity ...

  6. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Science.gov (United States)

    Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

    2017-01-01

    Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

  7. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  8. Anti-antimicrobial Peptides

    Science.gov (United States)

    Ryan, Lloyd; Lamarre, Baptiste; Diu, Ting; Ravi, Jascindra; Judge, Peter J.; Temple, Adam; Carr, Matthew; Cerasoli, Eleonora; Su, Bo; Jenkinson, Howard F.; Martyna, Glenn; Crain, Jason; Watts, Anthony; Ryadnov, Maxim G.

    2013-01-01

    Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance. PMID:23737519

  9. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  10. The production of Multiple Small Peptaibol Families by Single 14-Module Peptide Synthetases in Trichoderma/Hypocrea

    Energy Technology Data Exchange (ETDEWEB)

    Degenkolb, Thomas; Aghchehb, Razieh Karimi; Dieckmann, Ralf; Neuhof, Torsten; Baker, Scott E.; Druzhinina, Irina S.; Kubicek, Christian P.; Brückner, Hans; von Dohren, Hans

    2012-03-01

    The most common peptaibibiotic structures are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of 3 Trichoderma strains of the major clades reveal the presence of up to 3 types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid adding modules. We here provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina) and T. atroviride produces both 11- and 14- residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid activating domains and modules. The structures of these peptides may be predicted from the gene structures and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 new sequences), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.

  11. Antimicrobial Peptides in Echinoderms

    OpenAIRE

    Li, C; Haug, T; K Stensvåg

    2010-01-01

    Antimicrobial peptides (AMPs) are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. Here we summarize the knowledge of such peptides in echinoderms. Strongylocins are a novel family of cysteine-rich AMPs, recently identified in the sea urchins, Strongylocentrotus droebachiensis and S. purpuratus. Although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, d...

  12. Immunotherapy with Allergen Peptides

    OpenAIRE

    Larché Mark

    2007-01-01

    Specific allergen immunotherapy (SIT) is disease-modifying and efficacious. However, the use of whole allergen preparations is associated with frequent allergic adverse events during treatment. Many novel approaches are being designed to reduce the allergenicity of immunotherapy preparations whilst maintaining immunogenicity. One approach is the use of short synthetic peptides which representing dominant T cell epitopes of the allergen. Short peptides exhibit markedly reduced capacity to cro...

  13. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    Directory of Open Access Journals (Sweden)

    Daniel Braga

    2016-12-01

    Full Text Available Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

  14. Torque generation mechanism of ATP synthase

    Science.gov (United States)

    Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

    2010-03-01

    ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

  15. Recent progress in physicochemical characteristics of antimicrobial peptides%抗菌肽理化性质的研究进展

    Institute of Scientific and Technical Information of China (English)

    陈武; 黎定军; 丁彦; 肖启明; 周清明

    2012-01-01

    Antimicrobial peptides (AMPs) comprise an important part of the innate immunity system of host organism and provide effective protection for the host against bacteria, fungi, protozoa and viruses. They are synthesized either by ribosomal or non-ribosomal peptide synthetase. Usually, AMPs are positively charged small molecular weight proteins and have both a hydrophobic and hydrophilic side that enables the molecule to enter the membrane lipid bilayer. In this review, recent discoveries on such physicochemical characteristics of AMPs as conformation, cationicity, hydrophobicity, amphipathicity etc.are discussed.%抗菌肽(antimicrobial peptides,AMPs)是生物先天免疫系统的重要组成部分,由核糖体或非核糖体肽合成酶合成,可协助宿主有效应对细菌、真菌、原生生物和病毒等病原生物的胁迫.AMPs具有相对分子质量小、两亲性结构和携带正电荷等理化性质.综述抗菌肽构象、电荷及阳离子度、疏水性与疏水力矩、两亲性及其他属性等方面的研究进展.

  16. A new pathway for heavy metal detoxification in animals. Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans.

    Science.gov (United States)

    Vatamaniuk, O K; Bucher, E A; Ward, J T; Rea, P A

    2001-06-15

    Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.

  17. Natriuretic Peptides, Diagnostic and Prognostic Biomarkers

    NARCIS (Netherlands)

    J.H.W. Rutten (Joost)

    2010-01-01

    textabstractIn humans, the natriuretic peptide family consists of three different types of peptides: atrial natriuretic peptide (synonym: atrial natriuretic factor), B-type natriuretic peptide (synonym: brain natriuretic peptide) and C-natriuretic peptide.1 Atrial natriuretic peptide (ANP) was the f

  18. Structure and activity of NO synthase inhibitors specific to the L-arginine binding site.

    Science.gov (United States)

    Proskuryakov, S Ya; Konoplyannikov, A G; Skvortsov, V G; Mandrugin, A A; Fedoseev, V M

    2005-01-01

    Synthesis of compounds containing a fragment similar to the guanidine group of L-arginine, which is a substrate of nitric oxide synthase (NOS), is the main direction in creating NOS inhibitors. The inhibitory effect of such compounds is caused not only by their competition with the substrate for the L-arginine-binding site and/or oxidizing center of the enzyme (heme) but also by interaction with peptide motifs of the enzyme that influence its dimerization, affinity for cofactors, and interaction with associated proteins. Structures, activities, and relative in vitro and in vivo specificities of various NOS inhibitors (amino acid and non-amino acid) with linear or cyclic structure and containing guanidine, amidine, or isothiuronium group are considered. These properties are mainly analyzed by comparison with effects of the inhibitors on the inducible NOS.

  19. Cloning, expression, purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Kandasamy Eniyan; Urmi Bajpai

    2015-01-01

    Objective:To clone, express and purify2-methylcitrate synthase(Rv1131) gene of Mycobacterium tuberculosis(M. tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 gene was amplified by polymerase chain reaction usingM. tuberculosisH37Rv genomicDNA and cloned into pGEM-T easy vector and sequenced. The gene was sub-cloned in pET28c vector, expressed inEscherichia coliBL21(E. coliBL21) (DE3) cells and the recombinant protein was identified byWestern blotting.The protein was purified usingNickel affinity chromatography and the structural characteristics like sub-cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools.Tertiary structure of the protein and phylogenetic analysis was also established byin silico analysis.Results:The expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti-HIS antibodies and the protein was purified from the soluble fraction.In silicoanalysis showed that the protein contains no signal peptide and transmembrane helices.Active site prediction showed that the protein has histidine and aspartic acid residues at242,281 &332 positions respectively.Phylogenetic analysis showed 100% homology withmajor mycobacterial species.Secondary structure predicts2-methylcitrate synthase contain51.9% alpha-helix,8.7% extended strand and39.4% random coils.Tertiary structure of the protein was also established.Conclusions:The enzyme2-methylcitrate synthase from M. tuberculosisH37Rv has been successfully expressed and purified.The purified protein will further be utilized to develop assay methods for screening new inhibitors.

  20. A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)(1).

    Science.gov (United States)

    Roberts, Eric; Roberts, Alison W

    2009-02-01

    The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes ("terminal complexes"), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU-1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High-efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full-length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N- and C-terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.

  1. Natriuretic Peptides, Diagnostic and Prognostic Biomarkers

    OpenAIRE

    Rutten, Joost

    2010-01-01

    textabstractIn humans, the natriuretic peptide family consists of three different types of peptides: atrial natriuretic peptide (synonym: atrial natriuretic factor), B-type natriuretic peptide (synonym: brain natriuretic peptide) and C-natriuretic peptide.1 Atrial natriuretic peptide (ANP) was the fi rst natriuretic peptide to be discovered and in humans ANP is predominantly formed in the cardiomyocytes of the atria.2 B-type natriuretic peptide (BNP) was fi rst discovered in porcine brain hen...

  2. Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

    Directory of Open Access Journals (Sweden)

    N. D. Seago

    1995-01-01

    Full Text Available We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE2 levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO and N-acetyl-D-glucosaminidase (NAG activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE2 and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.

  3. Heteroexpression of the wheat phytochelatin synthase gene (TaPCS1) in rice enhances cadmium sensitivity

    Institute of Scientific and Technical Information of China (English)

    Feijuan Wang; Zhubing Wang; Cheng Zhu

    2012-01-01

    Phytochelatin synthase (PCS) (EC 2.3.2.15) catalyzes the final step of phytochelatins (PCs) biosynthesis.PCs are a family of cysteine-rich thiol-reactive and heavy metalbinding peptides that play an important role in sequestration and detoxification of heavy metals in plants.Previous studies have indicated that plants that overexpressed PCS displayed contrasting phenotypes,ranging from enhanced cadmium (Cd) tolerance to Cd hypersensitivity in Arabidopsis thaliana.In this study,the wheat phytochelatin synthase gene,TaPCS1,was heteroexpressed in wildtype rice (Oryza sativa L.,cv.Zhonghua 11) to evaluate the relationship between synthesis of PCs and Cd tolerance in rice.Data showed that the heteroexpression of TaPCS1 in rice enhanced Cd sensitivity and significantly increased Cd accumulation in shoots,but not in roots.Additionally,the PCS line exhibited a much higher content of PCs and non-protein thiols (NPTs) in shoots.Prominent changes in NPT composition led to reduced glutathione pool depletion and higher Cd content in cell organelles in shoots,followed by higher oxidative stress,which might result in Cd sensitivity.Therefore,the heteroexpression of TaPCS1 in rice is capable of increasing Cd accumulation in rice shoots and enhancing Cd sensitivity.

  4. [Four cases of aldosterone synthase deficiency in childhood].

    Science.gov (United States)

    Collinet, E; Pelissier, P; Richard, O; Gay, C; Pugeat, M; Morel, Y; Stephan, J-L

    2012-11-01

    Neonatal salt-wasting syndromes are rare but potentially serious conditions. Isolated hypoaldosteronism is an autosomal recessive inherited disorder of terminal aldosterone synthesis, leading to selective aldosterone deficiency. Two different biochemical forms of this disease have been described, called aldosterone synthase deficiency or corticosterone methyl oxydase, types I and II. In type I, there is no aldosterone synthase activity and the 18 hydroxycorticosterone (18 OHB) level is low, whereas in type II, a residual activity of aldosterone synthase persists and 18 OHB is overproduced. We report on four patients with isolated hypoaldosteronism. In 2 of them, who were recently diagnosed with aldosterone synthase deficit, we discuss the symptoms and treatment. The 2 other patients are now adults. We discuss the long-term outcome, the quality of adult life, aldosterone synthase deficits, as well as the pathophysiology and molecular analysis.

  5. Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation.

    Science.gov (United States)

    Picault, N; Cazalé, A C; Beyly, A; Cuiné, S; Carrier, P; Luu, D T; Forestier, C; Peltier, G

    2006-11-01

    The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

  6. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles

    OpenAIRE

    Piel, Jörn

    2002-01-01

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were ...

  7. Electron transfer in peptides.

    Science.gov (United States)

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  8. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  9. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway.

    Science.gov (United States)

    Taura, Futoshi; Tanaka, Shinji; Taguchi, Chiho; Fukamizu, Tomohide; Tanaka, Hiroyuki; Shoyama, Yukihiro; Morimoto, Satoshi

    2009-06-18

    Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.

  10. Protective Effect of Wheat Peptides against Indomethacin-Induced Oxidative Stress in IEC-6 Cells

    Directory of Open Access Journals (Sweden)

    Hong Yin

    2014-01-01

    Full Text Available Recent studies have demonstrated that wheat peptides protected rats against non-steroidal anti-inflammatory drugs-induced small intestinal epithelial cells damage, but the mechanism of action is unclear. In the present study, an indomethacin-induced oxidative stress model was used to investigate the effect of wheat peptides on the nuclear factor-κB(NF-κB-inducible nitric oxide synthase-nitric oxide signal pathway in intestinal epithelial cells-6 cells. IEC-6 cells were treated with wheat peptides (0, 125, 500 and 2000 mg/L for 24 h, followed by 90 mg/L indomethacin for 12 h. Wheat peptides significantly attenuated the indomethacin-induced decrease in superoxide dismutase and glutathione peroxidase activity. Wheat peptides at 2000 mg/L markedly decreased the expression of the NF-κB in response to indomethacin-induced oxidative stress. This study demonstrated that the addition of wheat peptides to a culture medium significantly inhibited the indomethacin-induced release of malondialdehyde and nitrogen monoxide, and increased antioxidant enzyme activity in IEC-6 cells, thereby providing a possible explanation for the protective effect proposed for wheat peptides in the prevention of indomethacin-induced oxidative stress in small intestinal epithelial cells.

  11. Protective effect of wheat peptides against indomethacin-induced oxidative stress in IEC-6 cells.

    Science.gov (United States)

    Yin, Hong; Pan, Xingchang; Song, Zhixiu; Wang, Shaokang; Yang, Ligang; Sun, Guiju

    2014-01-29

    Recent studies have demonstrated that wheat peptides protected rats against non-steroidal anti-inflammatory drugs-induced small intestinal epithelial cells damage, but the mechanism of action is unclear. In the present study, an indomethacin-induced oxidative stress model was used to investigate the effect of wheat peptides on the nuclear factor-κB(NF-κB)-inducible nitric oxide synthase-nitric oxide signal pathway in intestinal epithelial cells-6 cells. IEC-6 cells were treated with wheat peptides (0, 125, 500 and 2000 mg/L) for 24 h, followed by 90 mg/L indomethacin for 12 h. Wheat peptides significantly attenuated the indomethacin-induced decrease in superoxide dismutase and glutathione peroxidase activity. Wheat peptides at 2000 mg/L markedly decreased the expression of the NF-κB in response to indomethacin-induced oxidative stress. This study demonstrated that the addition of wheat peptides to a culture medium significantly inhibited the indomethacin-induced release of malondialdehyde and nitrogen monoxide, and increased antioxidant enzyme activity in IEC-6 cells, thereby providing a possible explanation for the protective effect proposed for wheat peptides in the prevention of indomethacin-induced oxidative stress in small intestinal epithelial cells.

  12. Amyloid beta-peptide worsens cognitive impairment following cerebral ischemia-reperfusion injury*****

    Institute of Scientific and Technical Information of China (English)

    Bo Song; Qiang Ao; Ying Niu; Qin Shen; Huancong Zuo; Xiufang Zhang; Yandao Gong

    2013-01-01

    Amyloid β-peptide, a major component of senile plaques in Alzheimer’s disease, has been impli-cated in neuronal cel death and cognitive impairment. Recently, studies have shown that the pathogenesis of cerebral ischemia is closely linked with Alzheimer’s disease. In this study, a rat model of global cerebral ischemia-reperfusion injury was established via occlusion of four arteries;meanwhile, fibril ar amyloid β-peptide was injected into the rat lateral ventricle. The Morris water maze test and histological staining revealed that administration of amyloid β-peptide could further aggravate impairments to learning and memory and neuronal cel death in the hippocampus of rats subjected to cerebral ischemia-reperfusion injury. Western blot showed that phosphorylation of tau protein and the activity of glycogen synthase kinase 3β were significantly stronger in cerebral is-chemia-reperfusion injury rats subjected to amyloidβ-peptide administration than those undergoing cerebral ischemia-reperfusion or amyloidβ-peptide administration alone. Conversely, the activity of protein phosphatase 2A was remarkably reduced in rats with cerebral ischemia-reperfusion injury fol owing amyloidβ-peptide administration. These findings suggest that amyloidβ-peptide can po-tentiate tau phosphorylation induced by cerebral ischemia-reperfusion and thereby aggravate cog-nitive impairment.

  13. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  14. The nitric oxide synthase of mouse spermatozoa.

    Science.gov (United States)

    Herrero, M B; Goin, J C; Boquet, M; Canteros, M G; Franchi, A M; Perez Martinez, S; Polak, J M; Viggiano, J M; Gimeno, M A

    1997-07-01

    Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

  15. Endothelial nitric oxide synthase in the microcirculation.

    Science.gov (United States)

    Shu, Xiaohong; Keller, T C Stevenson; Begandt, Daniela; Butcher, Joshua T; Biwer, Lauren; Keller, Alexander S; Columbus, Linda; Isakson, Brant E

    2015-12-01

    Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

  16. A Single Amino Acid Substitution Converts Benzophenone Synthase into Phenylpyrone Synthase*

    Science.gov (United States)

    Klundt, Tim; Bocola, Marco; Lütge, Maren; Beuerle, Till; Liu, Benye; Beerhues, Ludger

    2009-01-01

    Benzophenone metabolism provides a number of plant natural products with fascinating chemical structures and intriguing pharmacological activities. Formation of the carbon skeleton of benzophenone derivatives from benzoyl-CoA and three molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a member of the superfamily of type III polyketide synthases. A point mutation in the active site cavity (T135L) transformed BPS into a functional phenylpyrone synthase (PPS). The dramatic change in both substrate and product specificities of BPS was rationalized by homology modeling. The mutation may open a new pocket that accommodates the phenyl moiety of the triketide intermediate but limits polyketide elongation to two reactions, resulting in phenylpyrone formation. 3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a poor substrate for PPS. The aryl moiety of the triketide intermediate may be trapped in the new pocket by hydrogen bond formation with the backbone, thereby acting as an inhibitor. PPS is a promising biotechnological tool for manipulating benzoate-primed biosynthetic pathways to produce novel compounds. PMID:19710020

  17. Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.

    Science.gov (United States)

    Chen, Mengbin; Chou, Wayne K W; Toyomasu, Tomonobu; Cane, David E; Christianson, David W

    2016-04-15

    Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

  18. Dicyclopropylmethyl peptide backbone protectant.

    Science.gov (United States)

    Carpino, Louis A; Nasr, Khaled; Abdel-Maksoud, Adel Ali; El-Faham, Ayman; Ionescu, Dumitru; Henklein, Peter; Wenschuh, Holger; Beyermann, Michael; Krause, Eberhard; Bienert, Michael

    2009-08-20

    The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

  19. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders

    2015-01-01

    Therapeutic vaccines aim to control chronic HIV infection and eliminate the need for lifelong antiretroviral therapy (ART). Therapeutic HIV vaccine is being pursued as part of a functional cure for HIV/AIDS. We have outlined a basic protocol for inducing new T cell immunity during chronic HIV-1...... infection directed to subdominant conserved HIV-1 epitopes restricted to frequent HLA supertypes. The rationale for selecting HIV peptides and adjuvants are provided. Peptide subunit vaccines are regarded as safe due to the simplicity, quality, purity, and low toxicity. The caveat is reduced immunogenicity...

  20. β-PEPTIDES CYCLOBUTANIQUES

    OpenAIRE

    Fernandez, Carlos

    2008-01-01

    The synthesis of β-amino acids, structural analogues of?-Amino acids, is an issue essential in the development of oligopeptides. A lot of work has been conducted on the behavior of β-peptide (sequence of β-amino acids) as well as peptides mixed (mixed β-and β- amino acids). As a result, the conformational preference of β-amino acids will induce the appearance of a three-dimensional structure of the oligopeptide ordered. Thus, several types of helices, sheets and elbows were observed in β-olig...

  1. Immunotherapy with Allergen Peptides

    Directory of Open Access Journals (Sweden)

    Larché Mark

    2007-06-01

    Full Text Available Specific allergen immunotherapy (SIT is disease-modifying and efficacious. However, the use of whole allergen preparations is associated with frequent allergic adverse events during treatment. Many novel approaches are being designed to reduce the allergenicity of immunotherapy preparations whilst maintaining immunogenicity. One approach is the use of short synthetic peptides which representing dominant T cell epitopes of the allergen. Short peptides exhibit markedly reduced capacity to cross link IgE and activate mast cells and basophils, due to lack of tertiary structure. Murine pre-clinical studies have established the feasibility of this approach and clinical studies are currently in progress in both allergic and autoimmune diseases.

  2. Invertebrate FMRFamide related peptides.

    Science.gov (United States)

    Krajniak, Kevin G

    2013-06-01

    In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

  3. Role of cysteine residues in pseudouridine synthases of different families.

    Science.gov (United States)

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  4. Triptan-induced enhancement of neuronal nitric oxide synthase in trigeminal ganglion dural afferents underlies increased responsiveness to potential migraine triggers.

    Science.gov (United States)

    De Felice, Milena; Ossipov, Michael H; Wang, Ruizhong; Dussor, Gregory; Lai, Josephine; Meng, Ian D; Chichorro, Juliana; Andrews, John S; Rakhit, Suman; Maddaford, Shawn; Dodick, David; Porreca, Frank

    2010-08-01

    Migraine is a common neurological disorder often treated with triptans. Triptan overuse can lead to increased frequency of headache in some patients, a phenomenon termed medication overuse headache. Previous preclinical studies have demonstrated that repeated or sustained triptan administration for several days can elicit persistent neural adaptations in trigeminal ganglion cells innervating the dura, prominently characterized by increased labelling of neuronal profiles for calcitonin gene related peptide. Additionally, triptan administration elicited a behavioural syndrome of enhanced sensitivity to surrogate triggers of migraine that was maintained for weeks following discontinuation of drug, a phenomenon termed 'triptan-induced latent sensitization'. Here, we demonstrate that triptan administration elicits a long-lasting increase in identified rat trigeminal dural afferents labelled for neuronal nitric oxide synthase in the trigeminal ganglion. Cutaneous allodynia observed during the period of triptan administration was reversed by NXN-323, a selective inhibitor of neuronal nitric oxide synthase. Additionally, neuronal nitric oxide synthase inhibition prevented environmental stress-induced hypersensitivity in the post-triptan administration period. Co-administration of NXN-323 with sumatriptan over several days prevented the expression of allodynia and enhanced sensitivity to stress observed following latent sensitization, but not the triptan-induced increased labelling of neuronal nitric oxide synthase in dural afferents. Triptan administration thus promotes increased expression of neuronal nitric oxide synthase in dural afferents, which is critical for enhanced sensitivity to environmental stress. These data provide a biological basis for increased frequency of headache following triptans and highlight the potential clinical utility of neuronal nitric oxide synthase inhibition in preventing or treating medication overuse headache.

  5. Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

    Science.gov (United States)

    Bañuelos, O; Casqueiro, J; Steidl, S; Gutiérrez, S; Brakhage, A; Martín, J F

    2002-01-01

    There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

  6. Arabidopsis Indole Synthase,a Homolog of Tryptophan Synthase Alpha,is an Enzyme Involved in the Trp-independent Indole-containing Metabolite Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Rui Zhang; Bing Wang; Jian Ouyang; Jiayang Li; Yonghong Wang

    2008-01-01

    The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.

  7. The Pseudouridine Synthases Proceed through a Glycal Intermediate.

    Science.gov (United States)

    Veerareddygari, Govardhan Reddy; Singh, Sanjay K; Mueller, Eugene G

    2016-06-29

    The pseudouridine synthases isomerize (U) in RNA to pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2'. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.

  8. Peroxisomal and mitochondrial citrate synthase in CAM plants.

    Science.gov (United States)

    Zafra, M F; Segovia, J L; Alejandre, M J; García-Peregrín, E

    1981-12-01

    Citrate synthase wa studied for the first time in peroxisomes and mitochondria of crassulacean acid metabolism plants. Cellular organelles were isolated from Agave americana leaves by sucrose density gradient centrifugation and characterized by the use of catalase and cytochrome oxidase as marker enzymes, respectively. 48,000 X g centrifugation caused the breakdown of the cellular organelles. The presence of a glyoxylate cycle enzyme (citrate synthase) and a glycollate pathway enzyme (catalase) in the same organelles, besides the absence of another glyoxalate cycle enzyme (malate synthase) is reported for the first time, suggesting that peroxisomal and glyoxysomal proteins are synthesized at the same time and housed in he same organelle.

  9. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    . An inefficient post-translational prohormone maturation will also affect the biology of the cardiac natriuretic peptide system. This review aims at summarizing the myocardial synthesis of natriuretic peptides focusing on B-type natriuretic peptide, where new data has disclosed cardiac myocytes as highly...

  10. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    Science.gov (United States)

    Dozier, Jonathan K; Distefano, Mark D

    2012-02-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  11. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

    Science.gov (United States)

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico

    2014-07-01

    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  12. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  13. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...... in the human duodenum, jejunum, ileum and colon by immunohistochemistry. PGH synthase immunoreactivity appeared to be similar in all segments of the intestine. Most smooth muscle cells seemed to contain PGH synthase; however, the reaction in the lamina muscularis mucosae was much stronger than...... in the longitudinal and circular muscle layers. Endothelial cells in capillaries and larger vessels showed a positive reaction. In addition, unidentified cells in subserosa, at the level of Auerbach's plexus and in the submucosa were stained. We concluded that the smooth muscle cells of the human gut has a rather...

  14. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    NARCIS (Netherlands)

    Banzhaf, M.; van den Berg van Saparoea, B.; Terrak, M.; Fraipont, C.; Egan, A.; Philippe, J.; Zapun, A.; Breukink, E.; Nguyen-Distèche, M.; den Blaauwen, T.; Vollmer, W.

    2012-01-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan

  15. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Directory of Open Access Journals (Sweden)

    Zulfiqar Ahmad

    Full Text Available We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  16. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth.

    Science.gov (United States)

    Ahmad, Zulfiqar; Laughlin, Thomas F; Kady, Ismail O

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.

  17. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... 1Department of Environmental Biotechnology, Bharathidasan University, ... script and UA cloning vector (QIAGEN PCR Cloning Kit) was used to clone ..... Expression of a Arabidopsis sucrose synthase gene indicates a role.

  18. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function

    OpenAIRE

    Haines, Ricci J.; Corbin, Karen D.; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C.

    2012-01-01

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regu...

  19. APD: the Antimicrobial Peptide Database.

    Science.gov (United States)

    Wang, Zhe; Wang, Guangshun

    2004-01-01

    An antimicrobial peptide database (APD) has been established based on an extensive literature search. It contains detailed information for 525 peptides (498 antibacterial, 155 antifungal, 28 antiviral and 18 antitumor). APD provides interactive interfaces for peptide query, prediction and design. It also provides statistical data for a select group of or all the peptides in the database. Peptide information can be searched using keywords such as peptide name, ID, length, net charge, hydrophobic percentage, key residue, unique sequence motif, structure and activity. APD is a useful tool for studying the structure-function relationship of antimicrobial peptides. The database can be accessed via a web-based browser at the URL: http://aps.unmc.edu/AP/main.html.

  20. Radiolabelled peptides for oncological diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Laverman, Peter; Boerman, Otto C.; Oyen, Wim J.G. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Sosabowski, Jane K. [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom)

    2012-02-15

    Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The {sup 111}In-labelled somatostatin analogue octreotide (OctreoScan trademark) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours. (orig.)

  1. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  2. Unordered structured of proinsulin C-peptide in aqueous solution and in the presence of lipid vesicles.

    Science.gov (United States)

    Henriksson, M; Shafqat, J; Liepinsh, E; Tally, M; Wahren, J; Jörnvall, H; Johansson, J

    2000-02-01

    Proinsulin C-peptide ameliorates renal and autonomic nerve function and increases skeletal muscle blood flow, oxygen uptake and glucose transport in patients with insulin-dependent diabetes mellitus. These effects have in part been ascribed to the stimulatory influence of C-peptide on Na+,K+-ATPase and endothelial nitric oxide synthase. To evaluate the capacity of C-peptide to insert into lipid bilayers and form ion channels, C-peptide secondary structure and membrane interactions were studied with circular dichroism spectroscopy and size exclusion chromatography. C-peptide is shown to lack a stable secondary structure, both when part of proinsulin and when free in aqueous solution, although the N-terminal third of the peptide exhibits an alpha-helical conformation in trifluoroethanol. Moreover, C-peptide remains disordered in the aqueous solvent in the presence of lipid vesicles, regardless of vesicle composition. In conclusion, C-peptide is unlikely to elicit physiological effects through stable conformation-dependent interactions with lipid membranes.

  3. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  4. Nitric Oxide Synthases in Heart Failure

    Science.gov (United States)

    Carnicer, Ricardo; Crabtree, Mark J.; Sivakumaran, Vidhya

    2013-01-01

    Abstract Significance: The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca2+ homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology. Recent Advances: Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease. Critical Issues: Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress. Future Directions: Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance. Antioxid. Redox Signal. 18, 1078–1099. PMID:22871241

  5. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  6. Linking pseudouridine synthases to growth, development and cell competition.

    Science.gov (United States)

    Tortoriello, Giuseppe; de Celis, José F; Furia, Maria

    2010-08-01

    Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA-like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl-encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

  7. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  8. Evolution of Anabaenopeptin Peptide Structural Variability in the Cyanobacterium Planktothrix

    Science.gov (United States)

    Entfellner, Elisabeth; Frei, Mark; Christiansen, Guntram; Deng, Li; Blom, Jochen; Kurmayer, Rainer

    2017-01-01

    Cyanobacteria are frequently involved in the formation of harmful algal blooms wherein, apart from the toxic microcystins, other groups of bioactive peptides are abundant as well, such as anabaenopeptins (APs). The APs are synthesized nonribosomally as cyclic hexapeptides with various amino acids at the exocyclic position. We investigated the presence and recombination of the AP synthesis gene cluster (apnA-E) through comparing 125 strains of the bloom-forming cyanobacterium Planktothrix spp., which were isolated from numerous shallow and deep water habitats in the temperate and tropical climatic zone. Ten ecologically divergent strains were purified and genome sequenced to compare their entire apnA-E gene cluster. In order to quantify apn gene distribution patterns, all the strains were investigated by PCR amplification of 2 kbp portions of the entire apn gene cluster without interruption. Within the 11 strains assigned to P. pseudagardhii, P. mougeotii, or P. tepida (Lineage 3), neither apnA-E genes nor remnants were observed. Within the P. agardhii/P. rubescens strains from shallow waters (Lineage 1, 52 strains), strains both carrying and lacking apn genes occurred, while among the strains lacking the apnA-E genes, the presence of the 5′end flanking region indicated a gene cluster deletion. Among the strains of the more derived deep water ecotype (Lineage 2, 62 strains), apnA-E genes were always present. A high similarity of apn genes of the genus Planktothrix when compared with strains of the genus Microcystis suggested its horizontal gene transfer during the speciation of P. agardhii/P. rubescens. Genetic analysis of the first (A1-) domain of the apnA gene, encoding synthesis of the exocyclic position of the AP molecule, revealed four genotype groups that corresponded with substrate activation. Groups of genotypes were either related to Arginine only, the coproduction of Arginine and Tyrosine or Arginine and Lysine, or even the coproduction of Arginine

  9. Avian host defense peptides.

    Science.gov (United States)

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  10. Antimicrobial peptides in Echinoderms

    Directory of Open Access Journals (Sweden)

    C Li

    2010-05-01

    Full Text Available Antimicrobial peptides (AMPs are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. Here we summarize the knowledge of such peptides in echinoderms. Strongylocins are a novel family of cysteine-rich AMPs, recently identified in the sea urchins, Strongylocentrotus droebachiensis and S. purpuratus. Although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, dissimilar to other known AMPs. A family of heterodimeric AMPs, named centrocins, are also present in S. droebachiensis. Lysozymes and fragments of larger proteins, such as beta-thymocins, actin, histone 2A and filamin A have also been shown to display antimicrobial activities in echinoderms. Future studies on AMPs should be aimed in revealing how echinoderms use these AMPs in the immune response against microbial pathogens.

  11. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  12. Mitochondrial N-formyl peptides cause airway contraction and lung neutrophil infiltration via formyl peptide receptor activation.

    Science.gov (United States)

    Wenceslau, Camilla Ferreira; Szasz, Theodora; McCarthy, Cameron G; Baban, Babak; NeSmith, Elizabeth; Webb, R Clinton

    2016-04-01

    Respiratory failure is a common characteristic of systemic inflammatory response syndrome (SIRS) and sepsis. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns (DAMPs). Mitochondrial N-formyl peptides (F-MITs) are DAMPs that share similarities with bacterial N-formylated peptides, and are potent immune system activators. Recently, we observed that hemorrhagic shock-induced increases in plasma levels of F-MITs associated with lung damage, and that antagonism of formyl peptide receptors (FPR) ameliorated hemorrhagic shock-induced lung injury in rats. Corroborating these data, in the present study, it was observed that F-MITs expression is higher in plasma samples from trauma patients with SIRS or sepsis when compared to control trauma group. Therefore, to better understand the role of F-MITs in the regulation of lung and airway function, we studied the hypothesis that F-MITs lead to airway contraction and lung inflammation. We observed that F-MITs induced concentration-dependent contraction in trachea, bronchi and bronchioles. However, pre-treatment with mast cells degranulator or FPR antagonist decreased this response. Finally, intratracheal challenge with F-MITs increased neutrophil elastase expression in lung and inducible nitric oxide synthase and cell division control protein 42 expression in all airway segments. These data suggest that F-MITs could be a putative target to treat respiratory failure in trauma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Peptides and Food Intake

    OpenAIRE

    Carmen Sobrino Crespo; Aranzazu Perianes Cachero; Lilian Puebla Jiménez; Vicente eBarrios; Eduardo eArilla

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the r...

  14. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    Science.gov (United States)

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  15. Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

    Directory of Open Access Journals (Sweden)

    Huan Zhao

    2017-03-01

    Full Text Available Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii. Squalene synthase (SQS and cycloartenol synthase (CAS are key enzymes in triterpenoid and steroid biosynthesis. In this study, full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction (RACE-PCR approach. The SgSQS cDNA has a 1254 bp open reading frame (ORF encoding 417 amino acids, and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids. Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal. Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues, suggesting that steroids and mogrosides are competitors for the same precursors in fruits. Combined in silico prediction and subcellular localization, experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton, and SgCAS was likely located in the nucleus or cytosol. These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S. grosvenorii, and may facilitate improvements in mogroside content in fruit by regulating gene expression.

  16. [C-peptide physiological effects].

    Science.gov (United States)

    Shpakov, A O; Granstrem, O K

    2013-02-01

    In the recent years there were numerous evidences that C-peptide, which was previously considered as a product of insulin biosynthesis, is one of the key regulators of physiological processes. C-peptide via heterotrimeric G(i/o) protein-coupled receptors activates a wide range of intracellular effector proteins and transcription factors and, thus, controls the inflammatory and neurotrophic processes, pain sensitivity, cognitive function, macro- and microcirculation, glomerular filtration. These effects of C-peptide are mainly expressed in its absolute or relative deficiency occurred in type 1 diabetes mellitus and they are less pronounced when the level of C-peptide is close to normal. Replacement therapy with C-peptide prevents many complications of type 1 diabetes, such as atherosclerosis, diabetic peripheral neuropathy, and nephropathy. C-peptide interacts with the insulin hexamer complexes and induces their dissociation and, as a result, regulates the functional activity of the insulin signaling system. At the same time, C-peptide at the concentrations above physiological may demonstrate pro-inflammatory effects on the endothelial cells and cause atherosclerotic changes in the vessels, which should be considered in the study of pathogenic mechanisms of complications of type 2 diabetes mellitus, where the level of C peptide is increased, as well as in the development of approaches for C-peptide application in clinic. This review is devoted contemporary achievements and unsolved problems in the study of C-peptide, as an important regulator of physiological and biochemical processes.

  17. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  18. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  19. Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase.

    Science.gov (United States)

    Singh, Shailendra; Lee, Wonkyu; Dasilva, Nancy A; Mulchandani, Ashok; Chen, Wilfred

    2008-02-01

    Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (gamma-Glu-Cys)(n) moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and gamma-glutamyl cysteine (gamma-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3Delta strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n = 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn(2+) enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.

  20. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  1. The Pig PeptideAtlas

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius; Sun, Zhi;

    2016-01-01

    underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within...... the veterinary proteomics domain, and this article demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM...

  2. Biodiscovery of aluminum binding peptides

    Science.gov (United States)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  3. Antitumor Peptides from Marine Organisms

    Directory of Open Access Journals (Sweden)

    Mi Sun

    2011-10-01

    Full Text Available The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.

  4. Solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Jensen, Knud Jørgen

    2013-01-01

    This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective.......This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective....

  5. Expressing an (E)-b-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid

    Institute of Scientific and Technical Information of China (English)

    Gen-Ping Wang; Xiu-Dao Yu; Jia Fan; Cheng-She Wang; Lan-Qin Xia

    2015-01-01

    (E)-b-Farnesene (EbF) synthase catalyses the production of EbF, which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids’ natural enemies. Many plants possess EbF synthase genes and can release EbF to repel aphids. In order to effectively recruit the plant-derived EbF synthase genes for aphid control, by using chloroplast transit peptide (CTP) of the small subunit of Rubisco (rbcS) from wheat (Triticum aestivum L.), we targeted AabFS1, an EbF synthase gene from sweet wormwood (Artemisia annua L.), to the chloroplast of tobacco to generate CTP þ AabFS1 transgenic lines. The CTP þ AabFS1 transgenic tobacco plants could emit EbF at a level up to 19.25 ng/day per g fresh tissues, 4–12 fold higher than the AabFS1 transgenic lines without chloroplast targeting. Furthermore, aphid/parasitoid behavioral bio-assays demonstrated that the CTP þ AabFS1 transgenic tobacco showed enhanced repellence to green peach aphid (Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae, thus affecting aphid infestation at two trophic levels. These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EbF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.

  6. Peptides and Food Intake

    Science.gov (United States)

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  7. Anticancer peptides from bacteria

    Directory of Open Access Journals (Sweden)

    Tomasz M. Karpiński

    2013-08-01

    Full Text Available Cancer is a leading cause of death in the world. The rapid development of medicine and pharmacology allows to create new and effective anticancer drugs. Among modern anticancer drugs are bacterial proteins. Until now has been shown anticancer activity among others azurin and exotoxin A from Pseudomonas aeruginosa, Pep27anal2 from Streptococcus pneumoniae, diphtheria toxin from Corynebacterium diphtheriae, and recently discovered Entap from Enterococcus sp. The study presents the current data regarding the properties, action and anticancer activity of listed peptides.

  8. Exploring Protein-Peptide Binding Specificity through Computational Peptide Screening.

    Directory of Open Access Journals (Sweden)

    Arnab Bhattacherjee

    2013-10-01

    Full Text Available The binding of short disordered peptide stretches to globular protein domains is important for a wide range of cellular processes, including signal transduction, protein transport, and immune response. The often promiscuous nature of these interactions and the conformational flexibility of the peptide chain, sometimes even when bound, make the binding specificity of this type of protein interaction a challenge to understand. Here we develop and test a Monte Carlo-based procedure for calculating protein-peptide binding thermodynamics for many sequences in a single run. The method explores both peptide sequence and conformational space simultaneously by simulating a joint probability distribution which, in particular, makes searching through peptide sequence space computationally efficient. To test our method, we apply it to 3 different peptide-binding protein domains and test its ability to capture the experimentally determined specificity profiles. Insight into the molecular underpinnings of the observed specificities is obtained by analyzing the peptide conformational ensembles of a large number of binding-competent sequences. We also explore the possibility of using our method to discover new peptide-binding pockets on protein structures.

  9. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  10. Properties of peroxisomal and mitochondrial citrate synthase from Agave americana.

    Science.gov (United States)

    Segovia, J L; Zafra, M F; Alejandre, M J; García-Peregrín, E

    1982-09-01

    Adenine nucleotides were tested as effectors of peroxisomal and mitochondrial citrate synthase from Agave americana leaves in the presence of different concentrations of acetyl-CoA and oxalacetate substrates. ATP inhibited both enzyme activities but with a different inhibition profile. 1.0-7.5 mM ADP did not inhibit the peroxisomal citrate synthase in the presence of high substrate concentrations, while the mitochondrial enzyme was strongly inhibited by 1.0 mM ADP in the same conditions. Likewise, a different pattern was obtained with AMP on both peroxisomal and mitochondrial activities. The rate of citrate formation as function of acetyl-CoA and oxalacetate concentration was also studied in both fractions. Maximal velocity was highest in the peroxisomal fraction, whether acetyl-CoA or oxalacetate were the variable substrates. These differences indicate that peroxisomal and mitochondrial citrate synthases seem to be two different isoenzymes.

  11. Development, characterization, and epitope mapping of a panel of twenty-four monoclonal antibodies specific for human inducible nitric oxide synthase.

    Science.gov (United States)

    Webber, Robert J; Rodriguez, John G; Webber, Douglas S; Dunnebacke, Thelma H

    2005-02-01

    A panel of monoclonal antibodies (MAbs) to human inducible nitric oxide synthase (hiNOS) has been developed. By isotype analysis of the MAbs cloned from the 24 different positive hybridomas, 13 were determined to be mouse IgG1, two were mouse IgG2a, two were mouse IgG2b, and the seven others were mouse IgM antibodies: all contained kappa light chains. The anti-hiNOS MAbs were initially characterized by ELISA, RIA, Western blot, and immunocytochemistry, and then they were epitope mapped using synthetic peptides and a three-step mapping procedure. In the first step, each of the 24 MAbs was tested by indirect ELISA for binding to 96 overlapping 18-amino acid-long peptides that span the entire 1153-amino acid length of hiNOS. Eight IgG class anti-hiNOS MAbs were found to bind to one of five different peptides. In the second step, a series of amino terminal and carboxyl terminal truncated peptides were synthesized for each of the five peptides to which one or more of the MAbs bound. Each of the eight anti-hiNOS MAbs was found to bind to the truncated peptides with a unique specificity that identified the amino acid segment involved in binding. The third step in the epitope mapping process utilized three series of overlapping 5-, 6-, 7-, 8-, and 9-amino acid-long peptides for each of these segments and identified the exact amino acids of hiNOS involved in antibody binding. Anti-hiNOS MAbs 2A1-F8, 2D2-B2, 21C10-1D10, and 24B10-2C7 were found to be especially useful in different immunoassays.

  12. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    Science.gov (United States)

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  13. Endogenous opioid peptides and epilepsy

    NARCIS (Netherlands)

    J. Haffmans (Judith)

    1985-01-01

    textabstractIn recent years a large number of pept:ides, many of which were originall.y characterized in non-neural tissues, have been reported to be present in the central nervous system ( CNS) . The detection of these peptides within the CNS has raised many questions regarding their source and mec

  14. Endocrine cells producing regulatory peptides.

    Science.gov (United States)

    Solcia, E; Usellini, L; Buffa, R; Rindi, G; Villani, L; Zampatti, C; Silini, E

    1987-07-15

    Recent data on the immunolocalization of regulatory peptides and related propeptide sequences in endocrine cells and tumors of the gastrointestinal tract, pancreas, lung, thyroid, pituitary (ACTH and opioids), adrenals and paraganglia have been revised and discussed. Gastrin, xenopsin, cholecystokinin (CCK), somatostatin, motilin, secretin, GIP (gastric inhibitory polypeptide), neurotensin, glicentin/glucagon-37 and PYY (peptide tyrosine tyrosine) are the main products of gastrointestinal endocrine cells; glucagon, CRF (corticotropin releasing factor), somatostatin, PP (pancreatic polypeptide) and GRF (growth hormone releasing factor), in addition to insulin, are produced in pancreatic islet cells; bombesin-related peptides are the main markers of pulmonary endocrine cells; calcitonin and CGRP (calcitonin gene-related peptide) occur in thyroid and extrathyroid C cells; ACTH and endorphins in anterior and intermediate lobe pituitary cells, alpha-MSH and CLIP (corticotropin-like intermediate lobe peptide) in intermediate lobe cells; met- and leu-enkephalins and related peptides in adrenal medullary and paraganglionic cells as well as in some gut (enterochromaffin) cells; NPY (neuropeptide Y) in adrenaline-type adrenal medullary cells, etc.. Both tissue-appropriate and tissue-inappropriate regulatory peptides are produced by endocrine tumours, with inappropriate peptides mostly produced by malignant tumours.

  15. Urinary Peptides in Rett Syndrome.

    Science.gov (United States)

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  16. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    Cardiac-derived peptide hormones were identified more than 25 years ago. An astonishing amount of clinical studies have established cardiac natriuretic peptides and their molecular precursors as useful markers of heart disease. In contrast to the clinical applications, the biogenesis of cardiac...

  17. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  18. Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

    Directory of Open Access Journals (Sweden)

    Yan Ping Lim

    2016-06-01

    Full Text Available Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

  19. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    OpenAIRE

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control...

  20. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  1. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  2. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  3. Co-expression of Arabidopsis thaliana phytochelatin synthase and Treponema denticola cysteine desulfhydrase for enhanced arsenic accumulation.

    Science.gov (United States)

    Tsai, Shen-Long; Singh, Shailendra; Dasilva, Nancy A; Chen, Wilfred

    2012-02-01

    Arsenic is one of the most hazardous pollutants found in aqueous environments and has been shown to be a carcinogen. Phytochelatins (PCs), which are cysteine-rich and thio-reactive peptides, have high binding affinities for various metals including arsenic. Previously, we demonstrated that genetically engineered Saccharomyces cerevisiae strains expressing phytochelatin synthase (AtPCS) produced PCs and accumulated arsenic. In an effort to further improve the overall accumulation of arsenic, cysteine desulfhydrase, an aminotransferase that converts cysteine into hydrogen sulfide under aerobic condition, was co-expressed in order to promote the formation of larger AsS complexes. Yeast cells producing both AtPCS and cysteine desulfhydrase showed a higher level of arsenic accumulation than a simple cumulative effect of expressing both enzymes, confirming the coordinated action of hydrogen sulfide and PCs in the overall bioaccumulation of arsenic.

  4. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    Science.gov (United States)

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

  5. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    Science.gov (United States)

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  6. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Science.gov (United States)

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-26

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.

  7. The Remarkable Character of Porphobilinogen Synthase.

    Science.gov (United States)

    Jaffe, Eileen K

    2016-11-15

    Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn(2+); a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb(2+). PBGSs that do not require the soft metal ion Zn(2+) at the active site instead are suspected of using the hard metal Mg(2+). The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg(2+) as an allosteric activator. Mg(2+) functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do

  8. Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta Var. esculenta).

    Science.gov (United States)

    Lin, Da-Gin; Jeang, Chii-Ling

    2005-10-01

    Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340 bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 +/- 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms.

  9. Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia; Sonke, Theo; Quaedflieg, Peter J.; Janssen, Dick B.

    2013-01-01

    Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzyma

  10. Cloning of Sorghum bicolor Chloroplast Transit Peptide (CTP) of 5-enolpyruvylshikimate-3-phosphate Synthase(EPSPS) and Its Functional Validation in Transgenic Maize(Zea mays)%高粱5-烯醇式丙酮酰莽草酸-3-磷酸合酶基因(EPSPS)叶绿体转运肽(CTP)的克隆及其在转基因玉米中的功能验证

    Institute of Scientific and Technical Information of China (English)

    赵海铭; 宋伟彬; 赖锦盛

    2013-01-01

    5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)催化莽草酸-3-磷酸(S3P)与磷酸烯醇式丙酮酸(PEP)合成5-烯醇式丙酮酰莽草酸-3-磷酸(EPSP),具有与草甘膦结合的活性位点,且结合后会抑制EPSPS的活性,在植物抗除草剂基因工程中具有重要的应用价值.为了培育抗草甘膦玉米(Zea mays),本研究通过对高粱(Sorghum bicolor)EPSPS基因的结构分析,克隆了该基因5'端的叶绿体转运肽(chloroplast transit peptide,CTP).将该转运肽与来源于农杆菌(Agrobacterium tumefaciens)菌株CP4 EPSPS基因整合,以Ubiquitin为启动子,35S polyA为终止子,构建表达载体,同时以不含有转运肽的CP4 EPSPS基因为对照,遗传转化玉米得到稳定转基因株系;用PCR、Southern blot、ELISA等方法检测转基因玉米CP4 EPSPS基因的表达量并对其进行草甘膦抗性检测,研究发现,不含转运肽的转化事件虽然CP4 EPSPS基因表达量与含有转运肽的基本一致但却不具有草甘膦抗性,而含有转运肽的转化事件则抗性明显.说明转运肽并不影响CP4 EPSPS基因的表达,但对转基因植株草甘膦抗性起着重要作用,说明本研究克隆的叶绿体转运肽能够正常行使其生物学功能.研究结果为利用EPSPS基因培育抗草甘膦作物提供了重要参考资料.

  11. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  12. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B;

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  13. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  14. Biosynthesis of polyketides by trans-AT polyketide synthases.

    Science.gov (United States)

    Piel, Jörn

    2010-07-01

    This review discusses the biosynthesis of natural products that are generated by trans-AT polyketide synthases, a family of catalytically versatile enzymes that have recently been recognized as one of the major group of proteins involved in the production of bioactive polyketides. 436 references are cited.

  15. Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

    Directory of Open Access Journals (Sweden)

    David O. Hooks

    2014-06-01

    Full Text Available Polyhydroxyalkanoate (PHA is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC. Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.

  16. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  17. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    Science.gov (United States)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2 was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s−1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s−1 μM−1 for TgTPS2. The kinetic parameters were in agreement with previously published data. PMID:27721652

  18. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    2010-01-01

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highl

  19. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    Science.gov (United States)

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  20. Cloning and expression pattern of chitin synthase (CHS) gene in ...

    African Journals Online (AJOL)

    USER

    2010-08-16

    Aug 16, 2010 ... African Journal of Biotechnology Vol. 9(33), pp. 5297-5308, 16 ... Chitin synthase (CHS) plays an important role in biosynthesis of chitin .... strand cDNA Synthesis kit, 5'/3' RACE kit and pMD18-T vector were purchased from ...

  1. Potential of phage-displayed peptide library technology to identify functional targeting peptides

    Science.gov (United States)

    Krumpe, Lauren RH; Mori, Toshiyuki

    2010-01-01

    Combinatorial peptide library technology is a valuable resource for drug discovery and development. Several peptide drugs developed through phage-displayed peptide library technology are presently in clinical trials and the authors envision that phage-displayed peptide library technology will assist in the discovery and development of many more. This review attempts to compile and summarize recent literature on targeting peptides developed through peptide library technology, with special emphasis on novel peptides with targeting capacity evaluated in vivo. PMID:20150977

  2. Radiopharmaceutical development of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

    Fani, Melpomeni; Maecke, Helmut R. [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany)

    2012-02-15

    Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as {sup 99m}Tc, M{sup 3+} radiometals ({sup 111}In, {sup 86/90}Y, {sup 177}Lu, {sup 67/68}Ga), {sup 64/67}Cu, {sup 18}F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin

  3. Towards an Understanding of the Function of the Phytochelatin Synthase of Schistosoma mansoni

    Science.gov (United States)

    Rigouin, Coraline; Nylin, Elyse; Cogswell, Alexis A.; Schaumlöffel, Dirk; Dobritzsch, Dirk; Williams, David L.

    2013-01-01

    Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis. PMID:23383357

  4. Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Coraline Rigouin

    Full Text Available Phytochelatin synthase (PCS is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH. In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis and peptidase (hydrolysis of GSH S-conjugates activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

  5. Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni.

    Science.gov (United States)

    Rigouin, Coraline; Nylin, Elyse; Cogswell, Alexis A; Schaumlöffel, Dirk; Dobritzsch, Dirk; Williams, David L

    2013-01-01

    Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

  6. Enzymatic properties of chorismate synthase isozymes of tomato (Lycopersicon esculentum Mill.).

    Science.gov (United States)

    Braun, M; Henstrand, J M; Görlach, J; Amrhein, N; Schmid, J

    1996-01-01

    Three plastidic chorismate synthase isozymes (CS1, CS2 and CS2 delta) of tomato were identified by isolation of the corresponding cDNAs. These three cDNAs are derived from only two genes (LeCS1 and LeCS2). This additional complexity results from differential splicing of the primary transcript of one of the genes (LeCS2) giving rise to two different transcripts (CS2 and CS2 delta transcripts). All three isozymes were individually expressed in Escherichia coli both as precursor proteins with N-terminal transit peptides and as mature proteins. Only the mature but not the precursor isozymes CS1 and CS2 were enzymatically active. The enzyme CS2 delta was unstable in E. coli. Both CS1 and CS2 were purified to near homogeneity and their enzymatic properties were analyzed. They differ substantially in their Km values for the substrate 5-enol-pyruvylshikimate 3-phosphate (11 and 80 microM for the mature forms of CS1 and CS2, respectively). The two isozymes appear to be active only as oligomers, and the potential physiological implications of this result are discussed.

  7. Phytoene Synthase Gene Cloning from Citrus sinensis Osbeck cv.Cara Cara and Its Prokaryotic Expression

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-cheng; TAO Neng-guo; TONG Zhu; DENG Xiu-xin

    2008-01-01

    Using the mRNA from the fruit of Cara Cara as the template,the cDNA of phytoene synthase(PSY)gene was amplified by reverse transcription polymerse chain reaction(RT-PCR).Sequence analysis indicated that the eDNA was of 1 520 bp,which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids.The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%,respectively.A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY.The mature forms of PSY included a transmembrane(TM) domain.The recombinant plasmid pET-CitPSY was constructed by subeloning the full coding sequence of PSY eDNA into pET-28(+).After transformation of E.coil BL21 and induced by 1 mmol L-1 isopropyl-a-D-thiogalacropyranoside(IPTG),the fusion protein(6×His-PSY)with 52 kD was produced at a high level by prokaryotic expression system.The results of Western blot demonstrated that the fusion protein(6xHis-PSY)could be recognized by anti-6×His monoclonal antibody.The study could establish a basis for molecular improvement of Citrus fruit colors.

  8. New vasoactive peptides in cirrhosis

    DEFF Research Database (Denmark)

    Kimer, Nina; Goetze, Jens Peter; Bendtsen, Flemming;

    2014-01-01

    BACKGROUND: Patients with cirrhosis have substantial circulatory imbalance between vasoconstrictive and vasodilating forces. The study of circulatory vasoactive peptides may provide important pathophysiological information. This study aimed to assess concentrations, organ extraction and relations...... to haemodynamic changes in the pro-peptides copeptin, proadrenomedullin and pro-atrial natriuretic peptide (proANP) in patients with cirrhosis. MATERIALS AND METHODS: Fifty-four cirrhotic patients and 15 controls were characterized haemodynamically during a liver vein catheterization. Copeptin, proadrenomedullin...... found no extraction of copeptin, proadrenomedullin or proANP over the liver. Copeptin correlated with portal pressure (R=0·50, P

  9. Next generation natriuretic peptide measurement

    DEFF Research Database (Denmark)

    Hunter, Ingrid; Goetze, Jens P

    2012-01-01

    Plasma measurement of natriuretic peptides is a "must" for clinical laboratories. For the next generation measurement, the unraveling of the molecular complexity of the peptides points toward a more qualitative assessment, as the posttranslational processing also changes with disease. Changes...... in the molecular heterogeneity could in itself contain valuable information of clinical status, and the time seems right for industry and dedicated researchers in the field to get together and discuss the next generation natriuretic peptide measurement. In such an environment, new strategies can be developed...

  10. Peptide primary messengers in plants

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The peptide primary messengers regulate embryonic development,cell growth and many other activities in animal cells. But recent evidence verified that peptide primary messengers are also involved in plant defense responses, the recognition between pollen and stigma and keep the balance between cell proliferation and differentiations in shoot apical meristems. Those results suggest that plants may actually make wide use of peptide primary messengers, both in embryonic development and late life when they rally their cells to defend against pathogens and insect pests. The recent advance in those aspects is reviewed.

  11. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  12. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice.

    Science.gov (United States)

    Tsutsui, Masato; Tanimoto, Akihide; Tamura, Masahito; Mukae, Hiroshi; Yanagihara, Nobuyuki; Shimokawa, Hiroaki; Otsuji, Yutaka

    2015-01-01

    Nitric oxide (NO) is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs), all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling), lung abnormalities (accelerated pulmonary fibrosis), and bone abnormalities (increased bone mineral density and bone turnover). These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  13. Significance of nitric oxide synthases: Lessons from triple nitric oxide synthases null mice

    Directory of Open Access Journals (Sweden)

    Masato Tsutsui

    2015-01-01

    Full Text Available Nitric oxide (NO is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs, all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling, lung abnormalities (accelerated pulmonary fibrosis, and bone abnormalities (increased bone mineral density and bone turnover. These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

  14. Targeting the Eph System with Peptides and Peptide Conjugates.

    Science.gov (United States)

    Riedl, Stefan J; Pasquale, Elena B

    2015-01-01

    Eph receptor tyrosine kinases and ephrin ligands constitute an important cell communication system that controls development, tissue homeostasis and many pathological processes. Various Eph receptors/ephrins are present in essentially all cell types and their expression is often dysregulated by injury and disease. Thus, the 14 Eph receptors are attracting increasing attention as a major class of potential drug targets. In particular, agents that bind to the extracellular ephrin-binding pocket of these receptors show promise for medical applications. This pocket comprises a broad and shallow groove surrounded by several flexible loops, which makes peptides particularly suitable to target it with high affinity and selectivity. Accordingly, a number of peptides that bind to Eph receptors with micromolar affinity have been identified using phage display and other approaches. These peptides are generally antagonists that inhibit ephrin binding and Eph receptor/ ephrin signaling, but some are agonists mimicking ephrin-induced Eph receptor activation. Importantly, some of the peptides are exquisitely selective for single Eph receptors. Most identified peptides are linear, but recently the considerable advantages of cyclic scaffolds have been recognized, particularly in light of potential optimization towards drug leads. To date, peptide improvements have yielded derivatives with low nanomolar Eph receptor binding affinity, high resistance to plasma proteases and/or long in vivo half-life, exemplifying the merits of peptides for Eph receptor targeting. Besides their modulation of Eph receptor/ephrin function, peptides can also serve to deliver conjugated imaging and therapeutic agents or various types of nanoparticles to tumors and other diseased tissues presenting target Eph receptors.

  15. Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, Michael J.; Callister, Stephen J.; Miletto, Marzia; Williams, Kenneth H.; Nicora, Carrie D.; Lovely, Derek R.; Long, Philip E.; Lipton, Mary S.

    2011-01-01

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the U.S. Department of Energy’s Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  16. Development of a biomarker for Geobacter activity and strain composition; proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).

    Science.gov (United States)

    Wilkins, Michael J; Callister, Stephen J; Miletto, Marzia; Williams, Kenneth H; Nicora, Carrie D; Lovley, Derek R; Long, Philip E; Lipton, Mary S

    2011-01-01

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  17. Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R.; Long, P.E.; Lipton, M.S.

    2010-02-15

    Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

  18. Screening of TACE Peptide Inhibitors from Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.

  19. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  20. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  1. Identification of Peptides Inhibiting Adhesion of Monocytes to the Injured Vascular Endothelial Cells through Phage-displaying Screening

    Institute of Scientific and Technical Information of China (English)

    Yu GUO; Jia ZHANG; Ji-Cheng WANG; Feng-Xiang YAN; Bing-Yang ZHU; Hong-Lin HUANG; Duan-Fang LIAO

    2005-01-01

    Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the "adsorption-elution-amplification"procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin- 1 and intercellular adhesion molecule- 1 (ICAM- 1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.

  2. Viral O-GalNAc peptide epitopes

    DEFF Research Database (Denmark)

    Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas

    2016-01-01

    on a novel three-step procedure that identifies any reactive viral O-glycosyl peptide epitope with respect to (i) relevant peptide sequence, (ii) the reactive glycoform out of several possible glycopeptide isomers of that peptide sequence, and (iii) possibly tolerated carbohydrate or peptide structural...

  3. Neoglycolipidation for modulating peptide properties

    DEFF Research Database (Denmark)

    van Witteloostuijn, Søren Blok

    The alarming increase in the prevalence of obesity and associated comorbidities such as type 2 diabetes emphasizes the urgent need for new drugs with both anorectic and antidiabetic eects. Several peptide hormones secreted from the gastrointestinal tract play an important role in the physiological...... regulation of appetite, food intake, and glucose homeostasis, and many of these peptides display a signicant potential for treatment of obesity and/or type 2 diabetes. This Ph.D. thesis describes three novel approaches for utilizing gut peptides as the starting point for developing obesity and diabetes drugs...... of food intake, which was enhanced compared to native NMU. Project II explored the design, synthesis, and characterization of neoglycolipidated analogs of glucagon-like peptide 1 (GLP-1). Neoglycolipidation reduced lipophilicity and maintained or even improved in vitro potency towards the GLP-1 receptor...

  4. Neoglycolipidation for modulating peptide properties

    DEFF Research Database (Denmark)

    van Witteloostuijn, Søren Blok

    regulation of appetite, food intake, and glucose homeostasis, and many of these peptides display a signicant potential for treatment of obesity and/or type 2 diabetes. This Ph.D. thesis describes three novel approaches for utilizing gut peptides as the starting point for developing obesity and diabetes drugs...... of this thesis contribute to emphasize the tremendous therapeutic potential of gut peptides for treatment of obesity and diabetes.......The alarming increase in the prevalence of obesity and associated comorbidities such as type 2 diabetes emphasizes the urgent need for new drugs with both anorectic and antidiabetic eects. Several peptide hormones secreted from the gastrointestinal tract play an important role in the physiological...

  5. Therapeutical Potential of Venom Peptides

    Directory of Open Access Journals (Sweden)

    İlker Kelle

    2006-01-01

    Full Text Available The term of pharmazooticals is known as a few amount of drugs derived from natural sources such as plants, venomous species of snakes, spiders, scorpions, frogs, lizards and cone snails. Peptide components of venoms are directed against wide variety of pharmacological targets such as ion channels and receptors. At the beginning, a number of these peptides have been used in experimental studies for defining the physiological, biochemical and immunological activities of organisms like mammalians. In recent studies, it has been shown that venom peptides can be valuable in treatment of acute and chronic pain, autoimmune and cardiovascular diseases, neurological disorders and chronic inflammatory and tumoral processes. Therefore particularly in clinical approaches, these peptide molecules or their synthetic analogues are considered as alternative agents that can be used instead of classical drugs for many clinical disorders due to their potent activity besides very few side effects.

  6. Antimicrobial peptides from Capsicum sp.

    African Journals Online (AJOL)

    ajl yemi

    2011-12-30

    Dec 30, 2011 ... pathogens, it is a challenge to sustain food production. *Corresponding ... Genetically modified plants (GMPs) resistance to plant pathogens are an .... tically developed peptides have been tested in topic treatments during ...

  7. Peptides and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  8. Antimicrobial peptides in crustaceans

    Directory of Open Access Journals (Sweden)

    RD Rosa

    2010-11-01

    Full Text Available Crustaceans are a large and diverse invertebrate animal group that mounts a complex and efficient innate immune response against a variety of microorganisms. The crustacean immune system is primarily related to cellular responses and the production and release of important immune effectors into the hemolymph. Antimicrobial proteins and/or peptides (AMPs are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, 15 distinct AMP families are currently recognized, although the great majority (14 families comes from members of the order Decapoda. Crustacean AMPs are generally cationic, gene-encoded molecules that are mainly produced by circulating immune-competent cells (hemocytes or are derived from unrelated proteins primarily involved in other biological functions. In this review, we tentatively classified the crustacean AMPs into four main groups based on their amino acid composition, structural features and multi-functionality. We also attempted to summarize the current knowledge on their implication both in an efficient response to microbial infections and in crustacean survival.

  9. Antimicrobial peptides in annelids

    Directory of Open Access Journals (Sweden)

    A Tasiemski

    2008-06-01

    Full Text Available Gene encoded antimicrobial peptides (AMPs are widely distributed among living organisms including plants, invertebrates and vertebrates. They constitute important effectors of the innate immune response by exerting multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis and immune induction. In invertebrates, most of the data describe the characterization and/or the function of AMPs in the numerically and economically most representative group which are arthropods. Annelids are among the first coelomates and are therefore of special phylogenetic interest. Compared to other invertebrate groups, data on annelid’s immunity reveal heavier emphasis on the cellular than on the humoral response suggesting that immune defense of annelids seems to be principally developed as cellular immunity.This paper gives an overview of the variety of AMPs identified in the three classes of annelids, i.e. polychaetes, oligochaetes and achaetes. Their functions, when they have been studied, in the humoral or cellular response of annelids are also mentioned.

  10. Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease

    Directory of Open Access Journals (Sweden)

    Nicholas P Clayton

    2014-01-01

    Full Text Available Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20 and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1 was identified and conjugated to a cell penetrating peptide (GS-PPMO to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

  11. Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture

    Science.gov (United States)

    2016-01-01

    The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D80DQFD and N218DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H218O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-β-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-2H2]FDP and (R)-[1-2H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were

  12. The peptide toxin amylosin of Bacillus amyloliquefaciens from moisture-damaged buildings is immunotoxic, induces potassium efflux from mammalian cells, and has antimicrobial activity.

    Science.gov (United States)

    Rasimus-Sahari, Stiina; Teplova, Vera V; Andersson, Maria A; Mikkola, Raimo; Kankkunen, Päivi; Matikainen, Sampsa; Gahmberg, Carl G; Andersson, Leif C; Salkinoja-Salonen, Mirja

    2015-04-01

    Amylosin, a heat-stable channel-forming non-ribosomally synthesized peptide toxin produced by strains of Bacillus amyloliquefaciens isolated from moisture-damaged buildings, is shown in this paper to have immunotoxic and cytotoxic effects on human cells as well as antagonistic effects on microbes. Human macrophages exposed to 50 ng of amylosin ml(-1) secreted high levels of cytokines interleukin-1β (IL-1β) and IL-18 within 2 h, indicating activation of the NLRP3 inflammasome, an integral part of the innate immune system. At the same exposure level, expression of IL-1β and IL-18 mRNA increased. Amylosin caused dose-dependent potassium ion efflux from all tested mammalian cells (human monocytes and keratinocytes and porcine sperm cells) at 1 to 2 μM exposure. Amylosin also inhibited the motility of porcine sperm cells and depolarized the mitochondria of human keratinocytes. Amylosin may thus trigger the activation of the NLRP3 inflammasome and subsequently cytokine release by causing potassium efflux from exposed cells. The results of this study indicate that exposure to amylosin activates the innate immune system, which could offer an explanation for the inflammatory symptoms experienced by occupants of moisture-damaged buildings. In addition, the amylosin-producing B. amyloliquefaciens inhibited the growth of both prokaryotic and eukaryotic indoor microbes, and purified amylosin also had an antimicrobial effect. These antimicrobial effects could make amylosin producers dominant and therefore significant causal agents of health problems in some moisture-damaged sites.

  13. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

    Science.gov (United States)

    Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

    2016-01-01

    This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

  14. Peptides and Food Intake

    Directory of Open Access Journals (Sweden)

    Carmen Sobrino Crespo

    2014-04-01

    Full Text Available Nutrients created by the digestion of food are proposed to active G protein coupled receptors on the luminal side of enteroendocrine cells e.g. the L-cell. This stimulates the release of gut hormones. Hormones released from the gut and adipose tissue play an important rol in the regulation of food intake and energy expenditure (1.Many circulating signals, including gut hormones, can influence the activity of the arcuate nucleus (ARC neurons directly, after passing across the median eminence. The ARC is adjacent to the median eminence, a circumventricular organ with fenestrated capillaries and hence an incomplete blood-brain barrier (2. The ARC of the hypothalamus is believed to play a crucial role in the regulation of food intake and energy homeostasis. The ARC contains two populations of neurons with opposing effect on food intake (3. Medially located orexigenic neurons (i.e those stimulating appetite express neuropeptide Y (NPY and agouti-related protein (AgRP (4-5. Anorexigenic neurons (i.e. those inhibiting appetite in the lateral ARC express alpha-melanocyte stimulating hormone (α-MSH derived from pro-opiomelanocortin (POMC and cocaine and amphetamine-regulated transcript (CART (6. The balance between activities of these neuronal circuits is critical to body weight regulation.In contrast, other peripheral signals influence the hypothalamus indirectly via afferent neuronal pathway and brainstem circuits. In this context gastrointestinal’s vagal afferents are activated by mechanoreceptors and chemoreceptors, and converge in the nucleus of the tractus solitaries (NTS of the brainstem. Neuronal projections from the NTS, in turn, carry signals to the hypotalamus (1, 7. Gut hormones also alter the activity of the ascending vagal pathway from the gut to the brainstem. In the cases of ghrelin and Peptide tyrosine tyrosine (PYY, there are evidences for both to have a direct action on the arcuate nucleus and an action via the vagus nerve a

  15. Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

    NARCIS (Netherlands)

    Wu, Bian; Wijma, Hein J.; Song, Lu; Rozeboom, Henriette J.; Poloni, Claudia; Tian, Yue; Arif, Muhammad I.; Nuijens, Timo; Quaedflieg, Peter J. L. M.; Szymanski, Wiktor; Feringa, Ben L.; Janssen, Dick B.

    2016-01-01

    The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explore

  16. Automated solid-phase peptide synthesis to obtain therapeutic peptides

    Directory of Open Access Journals (Sweden)

    Veronika Mäde

    2014-05-01

    Full Text Available The great versatility and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last years. In order to increase the drugability of these frequently unstable and rapidly cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide synthesis (SPPS offers a suitable technology to produce chemically engineered peptides. This review concentrates on the application of SPPS by Fmoc/t-Bu protecting-group strategy, which is most commonly used. Critical issues and suggestions for the synthesis are covered. The development of automated methods from conventional to essentially improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGylation are described as covalent conjugation methods, which can be applied by a combination of automated and manual synthesis approaches. The synthesis and application of SPPS is described for neuropeptide Y receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated synthesis technologies.

  17. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian;

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...... classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...

  18. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along with ...... was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 μM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data....

  19. Conservation of capa peptide-induced nitric oxide signalling in Diptera.

    Science.gov (United States)

    Pollock, Valerie P; McGettigan, James; Cabrero, Pablo; Maudlin, Ian M; Dow, Julian A T; Davies, Shireen-A

    2004-11-01

    In D. melanogaster Malpighian (renal) tubules, the capa peptides stimulate production of nitric oxide (NO) and guanosine 3', 5'-cyclic monophosphate (cGMP), resulting in increased fluid transport. The roles of NO synthase (NOS), NO and cGMP in capa peptide signalling were tested in several other insect species of medical relevance within the Diptera (Aedes aegypti, Anopheles stephensi and Glossina morsitans) and in one orthopteran out-group, Schistocerca gregaria. NOS immunoreactivity was detectable by immunocytochemistry in tubules from all species studied. D. melanogaster, A. aegypti and A. stephensi express NOS in only principal cells, whereas G. morsitans and S. gregaria show more general NOS expression in the tubule. Measurement of associated NOS activity (NADPH diaphorase) shows that both D. melanogaster capa-1 and the two capa peptides encoded in the A. gambiae genome, QGLVPFPRVamide (AngCAPA-QGL) and GPTVGLFAFPRVamide (AngCAPA-GPT), all stimulate NOS activity in D. melanogaster, A. aegypti, A. stephensi and G. morsitans tubules but not in S. gregaria. Furthermore, capa-stimulated NOS activity in all the Diptera was inhibited by the NOS inhibitor l-NAME. All capa peptides stimulate an increase in cGMP content across the dipteran species, but not in the orthopteran S. gregaria. Similarly, all capa peptides tested stimulate fluid secretion in D. melanogaster, A. aegypti, A. stephensi and G. morsitans tubules but are either without effect or are inhibitory on S. gregaria. Consistent with these results, the Drosophila capa receptor was shown to be expressed in Drosophila tubules, and its closest Anopheles homologue was shown to be expressed in Anopheles tubules. Thus, we provide the first demonstration of physiological roles for two putative A. gambiae neuropeptides. We also demonstrate neuropeptide modulation of fluid secretion in tsetse tubule for the first time. Finally, we show the generality of capa peptide action, to stimulate NO/cGMP signalling and

  20. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

  1. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC)

  2. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    expression analysis and proteomics have pointed to abnormalities in mitochondrial oxidative phosphorylation and cellular stress in muscle of type 2 diabetic subjects, and recent work suggests that impaired mitochondrial activity is another early defect in the pathogenesis of type 2 diabetes. This review...... will discuss the latest advances in the understanding of the molecular mechanisms underlying insulin resistance in human skeletal muscle in type 2 diabetes with focus on possible links between impaired glycogen synthase activity and mitochondrial dysfunction....

  3. Structure and Mechanistic Implications of a Tryptophan Synthase Quinonoid Intermediate

    Energy Technology Data Exchange (ETDEWEB)

    Barends,T.; Domratcheva, T.; Kulik, V.; Blumenstein, L.; Niks, D.; Dunn, M.; Schlichting, I.

    2008-01-01

    Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Whereas structures of other PLP-bound reaction intermediates have been determined, a high-quality structure of a quinonoid species has not been reported. We present the crystal structure of the indoline quinonoid intermediate of tryptophan synthase (see figure) and discuss its implications for the enzymatic mechanism and allosteric regulation.

  4. Reduced Expression of Lipoic Acid Synthase Accelerates Diabetic Nephropathy

    OpenAIRE

    Yi, Xianwen; Xu, Longquan; Hiller, Sylvia; Kim, Hyung-Suk; Nickeleit, Volker; James, Leighton R; Maeda, Nobuyo

    2011-01-01

    Oxidative stress contributes to the pathogenesis of diabetic nephropathy. In mitochondria, lipoic acid synthase produces α-lipoic acid, an antioxidant and an essential cofactor in α-ketoacid dehydrogenase complexes, which participate in glucose oxidation and ATP generation. Administration of lipoic acid abrogates diabetic nephropathy in animal models, but whether lower production of endogenous lipoic acid promotes diabetic nephropathy is unknown. Here, we crossed mice heterozygous for lipoic ...

  5. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  6. Perspectives and Peptides of the Next Generation

    Science.gov (United States)

    Brogden, Kim A.

    Shortly after their discovery, antimicrobial peptides from prokaryotes and eukaryotes were recognized as the next potential generation of pharmaceuticals to treat antibiotic-resistant bacterial infections and septic shock, to preserve food, or to sanitize surfaces. Initial research focused on identifying the spectrum of antimicrobial agents, determining the range of antimicrobial activities against bacterial, fungal, and viral pathogens, and assessing the antimicrobial activity of synthetic peptides versus their natural counterparts. Subsequent research then focused on the mechanisms of antimicrobial peptide activity in model membrane systems not only to identify the mechanisms of antimicrobial peptide activity in microorganisms but also to discern differences in cytotoxicity for prokaryotic and eukaryotic cells. Recent, contemporary work now focuses on current and future efforts to construct hybrid peptides, peptide congeners, stabilized peptides, peptide conjugates, and immobilized peptides for unique and specific applications to control the growth of microorganisms in vitro and in vivo.

  7. Peptides: A new class of anticancer drugs

    Directory of Open Access Journals (Sweden)

    Ryszard Smolarczyk

    2009-07-01

    Full Text Available Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides, which bind specifically to precise molecular targets located on the surface of tumor cells. Antibacterial peptides bind to both cell and mitochondrial membranes. Some of these peptides attach to the cell membrane, resulting in its disorganization. Other antibacterial peptides penetrate cancer cells without causing cell membrane damage, but they disrupt mitochondrial membranes. Thanks to phage and aptamer libraries, it has become possible to obtain synthetic peptides blocking or activating some target proteins found in cancer cells as well as in cells forming the tumor environment. These synthetic peptides can feature anti-angiogenic properties, block enzymes indispensable for sustained tumor growth, and reduce tumor ability to metastasize. In this review the properties of peptides belonging to both categories are discussed and attempts of their application for therapeutic purposes are outlined.

  8. Exploration of the Medicinal Peptide Space.

    Science.gov (United States)

    Gevaert, Bert; Stalmans, Sofie; Wynendaele, Evelien; Taevernier, Lien; Bracke, Nathalie; D'Hondt, Matthias; De Spiegeleer, Bart

    2016-01-01

    The chemical properties of peptide medicines, known as the 'medicinal peptide space' is considered a multi-dimensional subset of the global peptide space, where each dimension represents a chemical descriptor. These descriptors can be linked to biofunctional, medicinal properties to varying degrees. Knowledge of this space can increase the efficiency of the peptide-drug discovery and development process, as well as advance our understanding and classification of peptide medicines. For 245 peptide drugs, already available on the market or in clinical development, multivariate dataexploration was performed using peptide relevant physicochemical descriptors, their specific peptidedrug target and their clinical use. Our retrospective analysis indicates that clusters in the medicinal peptide space are located in a relatively narrow range of the physicochemical space: dense and empty regions were found, which can be explored for the discovery of novel peptide drugs.

  9. From bacterial to human dihydrouridine synthase: automated structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  10. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  11. Rotation and structure of FoF1-ATP synthase.

    Science.gov (United States)

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background.

  12. The pseudouridine synthases: revisiting a mechanism that seemed settled.

    Science.gov (United States)

    Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2004-10-13

    RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.

  13. Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

    Science.gov (United States)

    Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

    2016-02-23

    Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

  14. Phytochelatin synthase activity as a marker of metal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Adam, Vojtech [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Zehnalek, Josef; Beklova, Miroslava [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kizek, Rene, E-mail: kizek@sci.muni.cz [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic)

    2011-08-30

    Highlights: {yields} New tool for determination of phytochelatin synthase activity. {yields} The optimization of experimental condition for determination of the enzyme activity. {yields} First evaluation of K{sub m} for the enzyme. {yields} The effects of cadmium (II) not only on the activity of the enzyme but also on K{sub m}. -- Abstract: The synthesis of phytochelatins is catalyzed by {gamma}-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO{sub 3}){sub 2} for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 {sup o}C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K{sub m} for PCS was estimated as 2.3 mM.

  15. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    Science.gov (United States)

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  16. Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase.

    Science.gov (United States)

    Carman, George M; Han, Gil-Soo

    2017-08-16

    The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. The structural basis of Erwinia rhapontici isomaltulose synthase.

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop(330-339) in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

  18. Multi-substrate terpene synthases: their occurrence and physiological significance

    Directory of Open Access Journals (Sweden)

    Leila Pazouki

    2016-07-01

    Full Text Available Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15, and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5, mono- (C10 and diterpenes (C20. Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

  19. Peptide synthesis using unprotected peptides through orthogonal coupling methods.

    Science.gov (United States)

    Tam, J P; Lu, Y A; Liu, C F; Shao, J

    1995-01-01

    We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency. Images Fig. 1 PMID:8618926

  20. Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur–arene interaction as key to catalysis

    OpenAIRE

    Sun, Shi-jing; Horikawa, Yoshiki; Wada, Masahisa; SUGIYAMA, Junji; Imai, Tomoya

    2016-01-01

    Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherich...

  1. Twilight reloaded: the peptide experience

    Science.gov (United States)

    Weichenberger, Christian X.; Pozharski, Edwin; Rupp, Bernhard

    2017-01-01

    The de facto commoditization of biomolecular crystallography as a result of almost disruptive instrumentation automation and continuing improvement of software allows any sensibly trained structural biologist to conduct crystallo­graphic studies of biomolecules with reasonably valid outcomes: that is, models based on properly interpreted electron density. Robust validation has led to major mistakes in the protein part of structure models becoming rare, but some depositions of protein–peptide complex structure models, which generally carry significant interest to the scientific community, still contain erroneous models of the bound peptide ligand. Here, the protein small-molecule ligand validation tool Twilight is updated to include peptide ligands. (i) The primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) a new method used to score peptide-ligand models is presented; (iii) a few instructive and specific examples, including an electron-density-based analysis of peptide-ligand structures that do not contain any ligands, are discussed in detail; (iv) means to avoid such mistakes and the implications for database integrity are discussed and (v) some suggestions as to how journal editors could help to expunge errors from the Protein Data Bank are provided. PMID:28291756

  2. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Directory of Open Access Journals (Sweden)

    Hyun Jo Koo

    Full Text Available The essential oils of ginger (Zingiber officinale and turmeric (Curcuma longa contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+-germacrene D synthase and (S-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (--caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+-α-turmerone and (+-β-turmerone, are produced from (--α-zingiberene and (--β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.

  3. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

    Science.gov (United States)

    Shibuya, M; Zhang, H; Endo, A; Shishikura, K; Kushiro, T; Ebizuka, Y

    1999-11-01

    Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.

  4. Peptide-enhanced oral delivery of therapeutic peptides and proteins

    DEFF Research Database (Denmark)

    Kristensen, Mie; Foged, Camilla; Berthelsen, Jens;

    2013-01-01

    throughout the gastrointestinal (GI) tract, chemical stability is an inherent challenge when employing amino acid-based excipients for oral delivery, and multiple approaches have been investigated to improve this. The exact mechanisms of transepithelial translocation are discussed, and it is believed......Systemic therapy upon oral delivery of biologics, such as peptide and protein drugs is limited due to their large molecular size, their low enzymatic stability and their inability to cross the intestinal epithelium. Ways to overcome the epithelial barrier include the use of peptide-based excipients...

  5. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe Brandt; Von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  6. Intracellular peptides: From discovery to function

    Directory of Open Access Journals (Sweden)

    Emer S. Ferro

    2014-06-01

    Full Text Available Peptidomics techniques have identified hundreds of peptides that are derived from proteins present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular peptides to distinguish them from secretory pathway peptides that function primarily outside of the cell. The proteasome and thimet oligopeptidase participate in the production and metabolism of intracellular peptides. Many of the intracellular peptides are common among mouse tissues and human cell lines analyzed and likely to perform a variety of functions within cells. Demonstrated functions include the modulation of signal transduction, mitochondrial stress, and development; additional functions will likely be found for intracellular peptides.

  7. Recent development of peptide self-assembly

    Institute of Scientific and Technical Information of China (English)

    Xiubo Zhao; Fang Pan; Jian R. Lu

    2008-01-01

    Amino acids are the building blocks to build peptides and proteins. Recent development in peptide synthesis has however enabled us to mimic this natural process by preparing various long and short peptides possessing different conformations and biological functions. The self-assembly of short designed peptides into molecular nanostructures is becoming a growing interest in nanobiotechnology. Self-assembled peptides exhibit several attractive features for applications in tissue regeneration, drug delivery, biological surface engineering as well as in food science, cosmetic industry and antibiotics. The aim of this review is to introduce the readers to a number of representative studies on peptide self-assembly.

  8. Characterization of Synthetic Peptides by Mass Spectrometry

    DEFF Research Database (Denmark)

    Prabhala, Bala K; Mirza, Osman; Højrup, Peter;

    2015-01-01

    Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI-TOF-MS an......Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI...

  9. Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    Hui LU; Bing Zhu; Xin-Dong Xue

    2006-01-01

    AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue.METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (LPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined.RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury.CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

  10. Antiviral active peptide from oyster

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    An active peptide against herpes virus was isolated from the enzymic hydrolysate of oyster (Crassostrea gigas) and purified with the definite direction hydrolysis technique in the order of alcalase and bromelin. The hydrolysate was fractioned into four ranges of molecular weight (>10 kDa, 10-5 kDa, 5-1 kDa and <1 kDa) using ultrafiltration membranes and dialysis. The fraction of 10?5 kDa was purified using consecutive chromatographic methods including DEAE Sephadex A-25 column, Sephadex G-25 column, and high performance liquid chromatogram (HPLC) by activity-guided isolation. The antiviral effect of the obtained peptide on herpetic virus was investigated in Vero cells by observing cytopathic effect (CPE). The result shows that the peptide has high inhibitory activity on herpetic virus.

  11. NCAM Mimetic Peptides: An Update

    DEFF Research Database (Denmark)

    Berezin, Vladimir; Bock, Elisabeth

    2008-01-01

    pharmacological tools interfering with NCAM functions. Recent progress in our understanding of the structural basis of NCAM-mediated cell adhesion and signaling has allowed a structure-based design of NCAM mimetic peptides. Using this approach a number of peptides termed P2, P1-B, P-3-DE and P-3-G, whose...... sequences contain one or several NCAM homophilic binding sites involved in NCAM binding to itself, have been identified. By means of NMR titration analysis and molecular modeling a number of peptides derived from NCAM and targeting NCAM heterophilic ligands such as the fibroblast growth factor receptor...... in vitro and in vivo, making them attractive pharmacological tools suitable for drug development for the treatment of neurodegenerative disorders and impaired memory....

  12. CLUSEAN: a computer-based framework for the automated analysis of bacterial secondary metabolite biosynthetic gene clusters.

    Science.gov (United States)

    Weber, T; Rausch, C; Lopez, P; Hoof, I; Gaykova, V; Huson, D H; Wohlleben, W

    2009-03-10

    Bacterial secondary metabolites are an important source of antimicrobial and cytostatic drugs. These molecules are often synthesized in a stepwise fashion by multimodular megaenzymes that are encoded in clusters of genes encoding enzymes for precursor supply and modification. In this work,we present an open source software pipeline, CLUSEAN (CLUster SEquence ANalyzer) that helps to annotate and analyze such gene clusters. CLUSEAN integrates standard analysis tools, like BLAST and HMMer, with specific tools for the identification of the functional domains and motifs in nonribosomal peptide synthetases (NRPS)/type I polyketide synthases (PKS) and the prediction of specificities of NRPS.

  13. Peptide Antibiotics for ESKAPE Pathogens

    DEFF Research Database (Denmark)

    Thomsen, Thomas Thyge

    a cecropin-mellitin hybrid peptide and proved effective in killing colistin resistant Gram-negative A. baumannii in vitro. The molecule was improved with regard to toxicity, as measured by hemolytic ability. Further, this peptide is capable of specifically killing non-growing cells of colistin resistant A......Multi-drug resistance to antibiotics represents a global health challenge that results in increased morbidity and mortality rates. The annual death-toll is >700.000 people world-wide, rising to ~10 million by 2050. New antibiotics are lacking, and few are under development as return on investment...

  14. Novel Formulations for Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Ana Maria Carmona-Ribeiro

    2014-10-01

    Full Text Available Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy.

  15. Peptides and the new endocrinology

    Science.gov (United States)

    Schwyzer, Robert

    1982-01-01

    The discovery of regulatory peptides common to the nervous and the endocrine systems (brain, gut, and skin) has brought about a revolution in our concepts of endocrinology and neurology. We are beginning to understand some of the complex interrelationships between soma and psyche that might, someday, be important for an integrated treatment of diseases. Examples of the actions of certain peptides in the periphery and in the central nervous system are given, and their biosynthesis and molecular anatomy as carriers for information are discussed.

  16. Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora

    National Research Council Canada - National Science Library

    Nadine Ziemert; Anna Lechner; Matthias Wietz; Natalie Millán-Aguiñaga; Krystle L. Chavarria; Paul Robert Jensen

    2014-01-01

    .... Here we analyze genome sequence data derived from 75 strains of the marine actinomycete genus Salinispora for pathways associated with polyketide and nonribosomal peptide biosynthesis, the products...

  17. Antimicrobial properties of cultivable bacteria associated with seaweeds in the Gulf of Mannar on the southeast coast of India

    National Research Council Canada - National Science Library

    Chakraborty, K; Thilakan, B; Chakraborty, R.D

    2016-01-01

    .... Antimicrobial activity analysis combined with the results of amplifying genes encoding for polyketide synthetase and nonribosomal peptide synthetase showed that seaweed-associated bacteria had broad...

  18. An enhancer peptide for membrane-disrupting antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Zhang Hong

    2010-02-01

    Full Text Available Abstract Background NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4 by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn. Results In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against Staphylococcus aureus, whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P. Conclusions NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane.

  19. Self-assembled N-cadherin mimetic peptide hydrogels promote the chondrogenesis of mesenchymal stem cells through inhibition of canonical Wnt/β-catenin signaling.

    Science.gov (United States)

    Li, Rui; Xu, Jianbin; Wong, Dexter Siu Hong; Li, Jinming; Zhao, Pengchao; Bian, Liming

    2017-11-01

    N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Automated genome mining for natural products

    Directory of Open Access Journals (Sweden)

    Zajkowski James

    2009-06-01

    Full Text Available Abstract Background Discovery of new medicinal agents from natural sources has largely been an adventitious process based on screening of plant and microbial extracts combined with bioassay-guided identification and natural product structure elucidation. Increasingly rapid and more cost-effective genome sequencing technologies coupled with advanced computational power have converged to transform this trend toward a more rational and predictive pursuit. Results We have developed a rapid method of scanning genome sequences for multiple polyketide, nonribosomal peptide, and mixed combination natural products with output in a text format that can be readily converted to two and three dimensional structures using conventional software. Our open-source and web-based program can assemble various small molecules composed of twenty standard amino acids and twenty two other chain-elongation intermediates used in nonribosomal peptide systems, and four acyl-CoA extender units incorporated into polyketides by reading a hidden Markov model of DNA. This process evaluates and selects the substrate specificities along the assembly line of nonribosomal synthetases and modular polyketide synthases. Conclusion Using this approach we have predicted the structures of natural products from a diverse range of bacteria based on a limited number of signature sequences. In accelerating direct DNA to metabolomic analysis, this method bridges the interface between chemists and biologists and enables rapid scanning for compounds with potential therapeutic value.

  1. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors...... such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist...

  2. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  3. Part 1. Antimicrobial and Immunomodulatory Peptides

    African Journals Online (AJOL)

    Furthermore, some peptides have been shown to have mineral ... immunopotentiating and antimicrobial properties including .... that this will give a clarion call to focus on the benefits ..... peptide could also be used in cosmetic, eye-care, oral.

  4. 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

    OpenAIRE

    Bunney, Tom D.; van Walraven, Hendrika S.; de Boer, Albertus H.

    2001-01-01

    Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our und...

  5. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  6. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    Cranial parasympathetic outflow is mediated through the sphenopalatine ganglion (SPG). The present study was performed to examine the expression of the parasympathetic signaling transmitters and their receptors in human and rat SPG. Indirect immunofluorescence technique was used for the demonstra...

  7. Recent advances in solid phase peptide synthesis

    OpenAIRE

    White, P.D.

    2016-01-01

    Since its introduction by Merrifield half a century ago, solid phase peptide synthesis has evolved to become the enabling technology for the development of peptide therapeutics. Using modern methods, 100 - 1000s of peptides can be routinely synthesised in parallel for screening as leads for drug development and peptide APIs are produced in ton scale. In this talk I consider the state of art and report on recent advances to overcome remaining issues such as aspartimide formation, racemisation ...

  8. Development and use of engineered peptide deformylase in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia

    2012-01-01

    Deze thesis beschrijft het onderzoek naar potentieel van het gebruik van het peptide deformylase (PDF) in chemo enzymatische peptide synthese. PDF is geschikt voor selective N terminale deformylatie van bepaalde N-formyl-peptides zonder gelijktijdige hydrolyse van de peptide binding. Door de uitdagi

  9. Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer-nucleotide coacervate droplets.

    Science.gov (United States)

    Crosby, John; Treadwell, Tom; Hammerton, Michelle; Vasilakis, Konstantinos; Crump, Matthew P; Williams, David S; Mann, Stephen

    2012-12-18

    Compartmentalization of the minimal complex of actinorhodin polyketide synthase in coacervate liquid droplets produces enhanced yields of shunt polyketides under conditions of low and high ionic strength.

  10. Gene identification and functional analysis of methylcitrate synthase in citric acid-producing Aspergillus niger WU-2223L.

    Science.gov (United States)

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2013-01-01

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

  11. Water drives peptide conformational transitions

    CERN Document Server

    Nerukh, Dmitry

    2011-01-01

    Transitions between metastable conformations of a dipeptide are investigated using classical molecular dynamics simulation with explicit water molecules. The distribution of the surrounding water at different moments before the transitions and the dynamical correlations of water with the peptide's configurational motions indicate that water is the main driving force of the conformational changes.

  12. Glucagon-like peptide-1

    DEFF Research Database (Denmark)

    Deacon, C F; Holst, Jens Juul; Carr, R D

    1999-01-01

    Type 2 diabetes mellitus is a metabolic disease resulting in raised blood sugar which, if not satisfactorily controlled, can cause severe and often debilitating complications. Unfortunately, for many patients, the existing therapies do not give adequate control. Glucagon-like peptide-1 (GLP-1) is...

  13. Glucagon-like peptide-1

    DEFF Research Database (Denmark)

    Holst, Jens Juul

    2006-01-01

    The incretin hormones are intestinal polypeptides that enhance postprandial insulin secretion. Gastric inhibitory polypeptide (GIP) was initially thought to regulate gastric acid secretion, whereas glucagon-like peptide-1 (GLP-1) was discovered as a result of a systematic search for intestinal...

  14. The evolution of peptide hormones.

    Science.gov (United States)

    Niall, H D

    1982-01-01

    Despite limitations in our present knowledge it is already possible to discern the main features of peptide hormone evolution, since the same mechanisms (and indeed the same hormone molecules) function in many different ways. This underlying unity of organization has its basis in the tendency of biochemical networks, once established, to survive and diversify. The most surprising recent findings in endocrinology have been the discovery of vertebrate peptide hormones in multiple sites within the same organism, and the reports, persuasive but requiring confirmation, of vertebrate hormones in primitive unicellular organisms (20, 20a). Perhaps the major challenge for the future is to define the roles and interactions of the many peptide hormones identified in brain (18). The most primitive bacteria and the human brain, though an enormous evolutionary distance apart, may have more in common than we have recognized until now. As Axelrod & Hamilton have pointed out in a recent provocative article, "The Evolution of Cooperation" (1), bacteria, though lacking a brain, are capable of adaptive behavior that can be analysed in terms of game theory. It is clear that we can learn a great deal about the whole evolutionary process from a study of the versatile and durable peptide hormones molecules.

  15. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  16. Peptides and metallic nanoparticles for biomedical applications.

    NARCIS (Netherlands)

    Kogan, M.J.; Olmedo, I.; Hosta, L.; Guerrero, A.R.; Cruz Ricondo, L.J.; Albericio, F.

    2007-01-01

    In this review, we describe the contribution of peptides to the biomedical applications of metallic nanoparticles. We also discuss strategies for the preparation of peptide-nanoparticle conjugates and the synthesis of the peptides and metallic nanoparticles. An overview of the techniques used for th

  17. Toxins and antimicrobial peptides: interactions with membranes

    Science.gov (United States)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

  18. Diverse CLE peptides from cyst nematode species

    Science.gov (United States)

    Plant CLAVATA3/ESR (CLE)-like peptides play diverse roles in plant growth and development including maintenance of the stem cell population in the root meristem. Small secreted peptides sharing similarity to plant CLE signaling peptides have been isolated from several cyst nematode species including...

  19. Activity of Cathelicidin Peptides against Chlamydia spp.

    Science.gov (United States)

    Donati, Manuela; Di Leo, Korinne; Benincasa, Monica; Cavrini, Francesca; Accardo, Silvia; Moroni, Alessandra; Gennaro, Renato; Cevenini, Roberto

    2005-01-01

    The in vitro activity of six cathelicidin peptides against 25 strains of Chlamydia was investigated. SMAP-29 proved to be the most active peptide, reducing the inclusion numbers of all 10 strains of Chlamydia trachomatis tested by ≥50% at 10 μg/ml. This peptide was also active against C. pneumoniae and C. felis. PMID:15728927

  20. Single-molecule studies on individual peptides and peptide assemblies on surfaces.

    Science.gov (United States)

    Yang, Yanlian; Wang, Chen

    2013-10-13

    This review is intended to reflect the recent progress in single-molecule studies of individual peptides and peptide assemblies on surfaces. The structures and the mechanism of peptide assembly are discussed in detail. The contents include the following topics: structural analysis of single peptide molecules, adsorption and assembly of peptides on surfaces, folding structures of the amyloid peptides, interaction between amyloid peptides and dye or drug molecules, and modulation of peptide assemblies by small molecules. The explorations of peptide adsorption and assembly will benefit the understanding of the mechanisms for protein-protein interactions, protein-drug interactions and the pathogenesis of amyloidoses. The investigations on peptide assembly and its modulations could also provide a potential approach towards the treatment of the amyloidoses.

  1. 水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定%Isolation of Rice EPSP Synthase cDNA and Its Sequence Analysis and Copy Number Determination

    Institute of Scientific and Technical Information of China (English)

    徐军望; 魏晓丽; 李旭刚; 陈蕾; 冯德江; 朱祯

    2002-01-01

    根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物,利用RT-PCR方法首次从水稻(Oryza sativa L. subsp. indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为1 585 bp的cDNA片段,它含有一个完整的开放读码框,编码511个氨基酸,包括444个氨基酸组成的成熟肽序列以及N端的67个氨基酸组成的叶绿体转运肽序列.成熟肽氨基酸序列对比表明,除真菌来源的EPSP合酶变异较大外,其他来源的EPSP合酶同源性较高,均在51%以上.而叶绿体转运肽氨基酸序列同源性较低.Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在.RT-PCR分析表明,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达,在叶片中表达量最高.%In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

  2. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  3. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...... on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing...

  4. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. © 2014 Wiley Periodicals, Inc.

  5. Argininosuccinate synthase as a novel biomarker for inflammatory conditions.

    Science.gov (United States)

    Cao, Mengde; George, Thomas J; Prima, Victor; Nelson, David; Svetlov, Stanislav

    2013-05-01

    Argininosuccinate synthase (ASS) plays an important role in regulating metabolic functions in mammals. We previously reported that hepatic ASS is released into circulation at very high concentrations in response to endotoxin and acute liver injury. We propose that ASS may serve as a novel biomarker for various inflammatory conditions. Our data showed that ASS accumulated in serum and urine of septic, obese or tumor mice in a condition-dependent fashion. Moreover, ASS significantly increased in urine within the first week after tumor cell implantation in mice which subsequently develop tumors. These results suggest that ASS is a novel biomarker increased upon diverse inflammatory conditions.

  6. Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase.

    Science.gov (United States)

    Girish, Tavarekere S; Sharma, Eshita; Gopal, B

    2008-08-20

    Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.

  7. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Marina eKorotkova

    2011-01-01

    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  8. Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

    OpenAIRE

    Yin Liu; Xiangru Lu; Fu-Li Xiang; Man Lu; Qingping Feng

    2013-01-01

    Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3(-/-) mice at postnatal day 0. Our data s...

  9. Peptide crosslinked micelles: a new strategy for the design and synthesis of peptide vaccines

    OpenAIRE

    Hao, Jihua; Kwissa, Marcin; Pulendran, Bali; Murthy, Niren

    2006-01-01

    This report presents a new and simple methodology for the synthesis of multicomponent peptide vaccines, named the peptide crosslinked micelles (PCMs). The PCMs are core shell micelles designed to deliver peptide antigens and immunostimulatory DNA to antigen-presenting cells (APCs). They are composed of immunostimulatory DNA, peptide antigen, and a thiopyridal derived poly(ethylene glycol)-polylysine block copolymer. The peptide antigen acts as a crosslinker in the PCM strategy, which allows t...

  10. Peptide array-based characterization and design of ZnO-high affinity peptides.

    Science.gov (United States)

    Okochi, Mina; Sugita, Tomoya; Furusawa, Seiji; Umetsu, Mitsuo; Adschiri, Tadafumi; Honda, Hiroyuki

    2010-08-15

    Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self-assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO-binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO-binding peptide, 125 spot-synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO-binding sites were found as 6-mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles.

  11. Fabrication of Odor Sensor Using Peptide

    Science.gov (United States)

    Hotokebuchi, Yuta; Hayashi, Kenshi; Toko, Kiyoshi; Chen, Ronggang; Ikezaki, Hidekazu

    We report fabrication of an odor sensor using peptides. Peptides were designed to acquire the specific reception for a target odor molecule. Au surface of the sensor electrode was coated by the designed peptide using the method of self assembled monolayers (SAMs). Functionalized Au surfaces by the peptides were confirmed by ellipsometry and cyclic voltammetry. The odorants of vanillin, phenethyl alcohol and hexanol were discriminated by QCM sensor with the peptide surface. Moreover, we verified specific interaction between amino acid (Trp) and vanillin by fluorescence assay.

  12. Brain natriuretic peptide measurement in pulmonary medicine.

    Science.gov (United States)

    Salerno, Daniel; Marik, Paul E

    2011-12-01

    Serum levels of natriuretic peptides are well established as important biomarkers in patients with cardiac disease. Less attention has been placed on the role of natriuretic peptides in patients with pulmonary conditions. In several well-defined groups of patients with pulmonary disease natriuretic peptides provide the clinician with clinically valuable information. A limitation of the interpretation of natriuretic peptides in pulmonary disease is the confounding effect of concurrent conditions such as heart failure, hypoxia, sepsis and renal failure. The present paper reviews the role of natriuretic peptides for diagnosis, risk stratification and prognosis of several pulmonary disorders.

  13. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  14. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Regulation of adenosine triphosphate-sensitive potassium channels suppresses the toxic effects of amyloid-beta peptide (25-35)

    Institute of Scientific and Technical Information of China (English)

    Min Kong; Maowen Ba; Hui Liang; Peng Shao; Tianxia Yu; Ying Wang

    2013-01-01

    In this study, we treated PC12 cells with 0-20 μM amyloid-β peptide (25-35) for 24 hours to induce cytotoxicity, and found that 5-20 μM amyloid-β peptide (25-35) decreased PC12 cell viability, but adenosine triphosphate-sensitive potassium channel activator diazoxide suppressed the decrease reactive oxygen species levels. These protective effects were reversed by the selective mitochondrial adenosine triphosphate-sensitive potassium channel blocker 5-hydroxydecanoate. An inducible nitric oxide synthase inhibitor, Nω-nitro-L-arginine, also protected PC12 cells from intracellular reactive oxygen species levels. However, the H2O2-degrading enzyme catalase could that the increases in both mitochondrial membrane potential and reactive oxygen species levels adenosine triphosphate-sensitive potassium channels and nitric oxide. Regulation of adenosine triphosphate-sensitive potassium channels suppresses PC12 cell cytotoxicity induced by amyloid-β

  16. Towards the MHC-peptide combinatorics.

    Science.gov (United States)

    Kangueane, P; Sakharkar, M K; Kolatkar, P R; Ren, E C

    2001-05-01

    The exponentially increased sequence information on major histocompatibility complex (MHC) alleles points to the existence of a high degree of polymorphism within them. To understand the functional consequences of MHC alleles, 36 nonredundant MHC-peptide complexes in the protein data bank (PDB) were examined. Induced fit molecular recognition patterns such as those in MHC-peptide complexes are governed by numerous rules. The 36 complexes were clustered into 19 subgroups based on allele specificity and peptide length. The subgroups were further analyzed for identifying common features in MHC-peptide binding pattern. The four major observations made during the investigation were: (1) the positional preference of peptide residues defined by percentage burial upon complex formation is shown for all the 19 subgroups and the burial profiles within entries in a given subgroup are found to be similar; (2) in class I specific 8- and 9-mer peptides, the fourth residue is consistently solvent exposed, however this observation is not consistent in class I specific 10-mer peptides; (3) an anchor-shift in positional preference is observed towards the C terminal as the peptide length increases in class II specific peptides; and (4) peptide backbone atoms are proportionately dominant at the MHC-peptide interface.

  17. Therapeutic uses of gastrointestinal peptides.

    Science.gov (United States)

    Redfern, J S; O'Dorisio, T M

    1993-12-01

    The GI tract is one of nature's great pharmacies. Most, if not all, biologically active peptides can be found there, and it is quite likely that others remain to be discovered. Our ability to exploit this resource has expanded considerably over the past two decades. Advances in analytical techniques have allowed investigators to rapidly isolate and purify new compounds from tissue extracts. Sequencing and de novo synthesis of newly discovered peptides are now routine, and the structural modifications required to alter activity and tailor a compound to a particular use are easily made. A number of gastrointestinal peptides or their analogues for use in clinical studies are available from commercial sources (see Table 7). Somatostatin is the first gut peptide to successfully complete development and yield a pharmaceutical compound with a broad range of action. Several of the peptides discussed in this article have similar potential. TRH stands out as a candidate because of its effectiveness in the treatment of experimental spinal cord injury and a variety of shock states. Such a broad range of action in critical fields may justify the intensive development required to yield potent, long-acting, and highly specific analogues. Similarly, the antimetastatic and immunostimulant properties of the enkephalins offer promise for new therapies in the treatment of AIDS, ARC, and cancer. Studies with amylin may lead to new and more precise regimens of blood sugar control in insulin-dependent diabetics and could in turn, prevent some of the worst long-term effects of the disease. The development of effective intranasal forms of GHRH could spare children with GH-GHRH deficiency the distress of repeated injections and help to prevent excessive GH blood levels. Secretin, glucagon, or CGRP might be used one day in cardiovascular emergencies, and VIP or its analogues could prove effective in the treatment of asthma. Although preliminary results with many of these peptides are

  18. The first salamander defensin antimicrobial peptide.

    Directory of Open Access Journals (Sweden)

    Ping Meng

    Full Text Available Antimicrobial peptides have been widely identified from amphibian skins except salamanders. A novel antimicrobial peptide (CFBD was isolated and characterized from skin secretions of the salamander, Cynops fudingensis. The cDNA encoding CFBD precursor was cloned from the skin cDNA library of C. fudingensis. The precursor was composed of three domains: signal peptide of 17 residues, mature peptide of 41 residues and intervening propeptide of 3 residues. There are six cysteines in the sequence of mature CFBD peptide, which possibly form three disulfide-bridges. CFBD showed antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli. This peptide could be classified into family of β-defensin based on its sequence similarity with β-defensins from other vertebrates. Evolution analysis indicated that CFBD was close to fish β-defensin. As far as we know, CFBD is the first β-defensin antimicrobial peptide from salamanders.

  19. Computer-Aided Design of Antimicrobial Peptides

    DEFF Research Database (Denmark)

    Fjell, Christopher D.; Hancock, Robert E.W.; Jenssen, Håvard

    2010-01-01

    chemical parameters with biological activities of the peptide, using statistical methods. In this review we will discuss two different in silico strategies of computer-aided antibacterial peptide design, a linear correlation model build as an extension of traditional principal component analysis (PCA......) and a non-linear artificial neural network model. Studies on structurally diverse peptides, have concluded that the PCA derived model are able to guide the antibacterial peptide design in a meaningful way, however requiring rather a high homology between the peptides in the test-set and the in silico...... library, to ensure a successful prediction. In contrast, the neural network model, though significantly less explored in relation to antimicrobial peptide design, has proven extremely promising, demonstrating impressive prediction success and ranking of random peptide libraries correlating well...

  20. Biology of the CAPA peptides in insects.

    Science.gov (United States)

    Predel, R; Wegener, C

    2006-11-01

    CAPA peptides have been isolated from a broad range of insect species as well as an arachnid, and can be grouped into the periviscerokinin and pyrokinin peptide families. In insects, CAPA peptides are the characteristic and most abundant neuropeptides in the abdominal neurohemal system. In many species, CAPA peptides exert potent myotropic effects on different muscles such as the heart. In others, including blood-sucking insects able to transmit serious diseases, CAPA peptides have strong diuretic or anti-diuretic effects and thus are potentially of medical importance. CAPA peptides undergo cell-type-specific sorting and packaging, and are the first insect neuropeptides shown to be differentially processed. In this review, we discuss the current knowledge on the structure, distribution, receptors and physiological actions of the CAPA peptides.

  1. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  2. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    Science.gov (United States)

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.

  3. Insulin transcriptionally regulates argininosuccinate synthase to maintain vascular endothelial function.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Meininger, Cynthia J; Eichler, Duane C

    2012-04-27

    Diminished vascular endothelial cell nitric oxide (NO) production is a major factor in the complex pathogenesis of diabetes mellitus. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate-limiting step of the citrulline-NO cycle. Using serum starved, cultured vascular endothelial cells, we show that insulin up-regulates AS and eNOS transcription to support NO production. Moreover, we show that insulin enhances NO production in response to physiological cues such as bradykinin. To translate these results to an in vivo model, we show that AS transcription is diminished in coronary endothelial cells isolated from rats with streptozotocin (STZ)-induced diabetes. Importantly, we demonstrate restoration of AS and eNOS transcription by insulin treatment in STZ-diabetic rats, and show that this restoration was accompanied by improved endothelial function as measured by endothelium-dependent vasorelaxation. Overall, this report demonstrates, both in cell culture and whole animal studies, that insulin maintains vascular function, in part, through the maintenance of AS transcription, thus ensuring an adequate supply of arginine to maintain vascular endothelial response to physiological cues. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  5. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing......CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...

  6. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    Directory of Open Access Journals (Sweden)

    Milan Hargovan

    2014-02-01

    Full Text Available The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors.

  7. Aldosterone synthase inhibitors in hypertension: current status and future possibilities.

    Science.gov (United States)

    Hargovan, Milan; Ferro, Albert

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors.

  8. Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

    Science.gov (United States)

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides. PMID:22235310

  9. IPC synthase as a useful target for antifungal drugs.

    Science.gov (United States)

    Sugimoto, Yuichi; Sakoh, Hiroki; Yamada, Koji

    2004-12-01

    Inositol phosphorylceramide (IPC) synthase is a common and essential enzyme in fungi and plants, which catalyzes the transfer of phosphoinositol to the C-1 hydroxy of ceramide to produce IPC. This reaction is a key step in fungal sphingolipid biosynthesis, therefore the enzyme is a potential target for the development of nontoxic therapeutic antifungal agents. Natural products with a desired biological activity, aureobasidin A (AbA), khafrefungin, and galbonolide A, have been reported. AbA, a cyclic depsipeptide containing 8 amino acids and a hydroxyl acid, is a broad spectrum antifungal with strong activity against many pathogenic fungi such as Candida spp., Cryptococcus neoformans, and some Aspergillus spp. Khafrefungin, an aldonic acid ester with a C22 long alkyl chain, has antifungal activity against C. albicans, Cr. Neoformans, and Saccharomyces cerevisiae. Galbonolide A is a 14-membered macrolide with fungicidal activity against clinically important strains, and is especially potent against Cr. neoformans. These classes of natural products are potent and specific antifungal agents. We review current progress in the development of IPC synthase inhibitors with antifungal activities, and present structure-activity relationships (SAR), physicochemical and structural properties, and synthetic methodology for chemical modification.

  10. Eugenol synthase genes in floral scent variation in Gymnadenia species.

    Science.gov (United States)

    Gupta, Alok K; Schauvinhold, Ines; Pichersky, Eran; Schiestl, Florian P

    2014-12-01

    Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of "floral scent genes" and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

  11. Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Lan HUANG; Dong-Yang LI; Shao-Xiao WANG; Shi-Ming ZHANG; Jun-Hui CHEN; Xiang-Fu WU

    2005-01-01

    Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.

  12. Tryptophan synthase of Phaeophyceae originated from the secondary host nucleus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yalan; CHI Shan; WU Shuangxiu; LIU Cui; YU Jun; WANG Xumin; CHEN Shengping; LIU Tao

    2014-01-01

    Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In pro-karyotes, tryptophan synthase is a multi-enzyme complex, and it consists ofαandβsubunit which forms anα-ββ-αcomplex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophy-ceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rho-dophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect withαsubunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.

  13. Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene.

    Science.gov (United States)

    Shaik, Abjal Pasha; Alsaeed, Abbas H; Sultana, Asma

    2012-01-01

    The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther's disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref NP_000366.1, NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14(th) Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.

  14. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  15. Anionic phospholipids modulate peptide insertion into membranes.

    Science.gov (United States)

    Liu, L P; Deber, C M

    1997-05-06

    While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which

  16. Experimental evidences of the NO action on a recombinant PrxII F from pea plant and its effect preventing the citrate synthase aggregation

    Directory of Open Access Journals (Sweden)

    Daymi Camejo

    2015-06-01

    Full Text Available S-nitrosylation is emerging as a key post-translational protein modification for the transduction of NO as a signaling molecule in plants. This data article supports the research article entitled “Functional and structural changes in plant mitochondrial PrxII F caused by NO” [1]. To identify the Cys residues of the recombinant PrxII F modified after the treatment with S-nitrosylating agents we performed the LC ESI–QTOF tandem MS and MALDI peptide mass fingerprinting analysis. Change in A650 nm was monitored to estimate the thermal aggregation of citrate synthase in the presence S-nitrosylated PrxII F. The effect of the temperature on the oligomerization pattern and aggregation of PrxII F was analysed by SDS-PAGE and changes in absorbance at 650 nm, respectively.

  17. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was ...

  18. Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

    Science.gov (United States)

    Del Campo, M; Kaya, Y; Ofengand, J

    2001-11-01

    There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

  19. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  20. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named

  1. KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery

    NARCIS (Netherlands)

    Mansoori Zangir, N.; Timmers, J.F.P.; Desprez, T.; Lessa Alvim Kamei, C.; Dees, D.C.T.; Vincken, J.P.; Visser, R.G.F.; Höfte, H.; Vernhettes, S.; Trindade, L.M.

    2014-01-01

    Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases.

  2. Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.

    NARCIS (Netherlands)

    Tamer, el M.K.; Lucker, J.; Bosch, D.; Verhoeven, H.A.; Verstappen, F.W.A.; Schwab, W.; Tunen, van A.J.; Voragen, A.G.J.; Maagd, de R.A.; Bouwmeester, H.J.

    2003-01-01

    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPIN

  3. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank;

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c...

  4. Peptide Antibiotics for ESKAPE Pathogens

    DEFF Research Database (Denmark)

    Thomsen, Thomas Thyge

    Multi-drug resistance to antibiotics represents a global health challenge that results in increased morbidity and mortality rates. The annual death-toll is >700.000 people world-wide, rising to ~10 million by 2050. New antibiotics are lacking, and few are under development as return on investment...... is considered poor compared to medicines for lifestyle diseases. According to the WHO we could be moving towards a post-antibiotic era in which previously treatable infections become fatal. Of special importance are multidrug resistant bacteria from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus...... and toxicity by utilizing of the fruit fly Drosophila melanogaster as a whole animal model. This was carried out by testing of antimicrobial peptides targeting Gram-positive bacteria exemplified by the important human pathogen methicillin resistant S. aureus (MRSA). The peptide BP214 was developed from...

  5. [Heterogenous expression of antimicrobial peptides].

    Science.gov (United States)

    Song, Shanshan; Hu, Guobin; Dong, Xianzhi

    2009-12-01

    Antimicrobial peptides (AMPs), a class of short proteins with a broad spectrum of antibacterial activities, are isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. They are a key component of the innate immune response in most multicellular organisms. Owing to their potent, broad-spectrum antibacterial activities and uneasy developing of drug resistance, these peptides are of great clinical significance. However, preparation of AMPs at a large scale is a severe challenge to the development of the commercial products. Undoubtedly, construction of high-level biological expression systems for the production of AMPs is the key in its clinical application process. Herein, we summarize the progress in researches on heterogenous expression of AMPs in prokaryotic expression systems and eukaryotic expression systems.

  6. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions

    DEFF Research Database (Denmark)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco

    2016-01-01

    . In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited...... for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions...... determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide...

  7. Encapsulation of bioactive whey peptides in soy lecithin-derived nanoliposomes: Influence of peptide molecular weight.

    Science.gov (United States)

    Mohan, Aishwarya; McClements, David Julian; Udenigwe, Chibuike C

    2016-12-15

    Encapsulation of peptides can be used to enhance their stability, delivery and bioavailability. This study focused on the effect of the molecular weight range of whey peptides on their encapsulation within soy lecithin-derived nanoliposomes. Peptide molecular weight did not have a major impact on encapsulation efficiency or liposome size. However, it influenced peptide distribution amongst the surface, core, and bilayer regions of the liposomes, as determined by electrical charge (ζ-potential) and FTIR analysis. The liposome ζ-potential depended on peptide molecular weight, suggesting that the peptide charged groups were in different locations relative to the liposome surfaces. FTIR analysis indicated that the least hydrophobic peptide fractions interacted more strongly with choline on the liposome surfaces. The results suggested that the peptides were unequally distributed within the liposomes, even at the same encapsulation efficiency. These findings are important for designing delivery systems for commercial production of encapsulated peptides with improved functional attributes.

  8. Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

    Science.gov (United States)

    Trier, Nicole Hartwig

    2015-01-01

    Characterization of peptide antibodies through identification of their target epitopes is of utmost importance. Understanding antibody specificity at the amino acid level provides the key to understand the specific interaction between antibodies and their epitopes and their use as research and diagnostic tools as well as therapeutic agents. This chapter describes a straightforward strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies: (1) overlapping peptides, used to locate antigenic regions; (2) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (3) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for fine mapping. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-sparing and straightforward approach for characterization of peptide antibodies.

  9. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions.

    Science.gov (United States)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco; Østergaard, Jesper

    2016-10-10

    Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations.

  10. Peptide Membranes in Chemical Evolution*

    OpenAIRE

    2009-01-01

    Simple surfactants achieve remarkable long-range order in aqueous environments. This organizing potential is seen most dramatically in biological membranes where phospholipid assemblies both define cell boundaries and provide a ubiquitous structural scaffold for controlling cellular chemistry. Here we consider simple peptides that also spontaneously assemble into exceptionally ordered scaffolds, and review early data suggesting that these structures maintain the functional diversity of protei...

  11. Antimicrobial peptides in human sepsis

    Directory of Open Access Journals (Sweden)

    Lukas eMartin

    2015-08-01

    Full Text Available Nearly 100 years ago, antimicrobial peptides (AMPs were identified as an important part of innate immunity. They exist in species from bacteria to mammals and can be isolated in body fluids and on surfaces constitutively or induced by inflammation. Defensins have anti-bacterial effects against Gram-positive and Gram-negative bacteria as well as anti-viral and anti-yeast effects. Human neutrophil peptides (HNP 1-3 and human beta-defensins (HBDs 1-3 are some of the most important defensins in humans. Recent studies have demonstrated higher levels of HNP -1-3 and HBD-2 in sepsis. The bactericidal/permeability increasing protein (BPI attenuates local inflammatory response and decreases systemic toxicity of endotoxins. Moreover, BPI might reflect the severity of organ dysfunction in sepsis. Elevated plasma lactoferrin is detected in patients with organ failure. HNP-1-3, lactoferrin, BPI and heparin-binding protein (HBP are increased in sepsis. Human lactoferrin peptide 1-11 (hLF 1-11 possesses antimicrobial activity and modulates inflammation. The recombinant form of lactoferrin (talactoferrin alpha, TLF has been shown to decrease mortality in critically ill patients. A phase II/III study with TLF in sepsis did not confirm this result. The growing number of multiresistant bacteria is an ongoing problem in sepsis therapy. Furthermore, antibiotics are known to promote the liberation of pro-inflammatory cell components and thus augment the severity of sepsis. Compared to antibiotics, AMPs kill bacteria but also neutralize pathogenic factors such as lipopolysaccharide (LPS. The obstacle to applying naturally occurring AMPs is their high nephro- and neurotoxicity. Therefore, the challenge is to develop peptides to treat septic patients effectively without causing harm. This overview focuses on natural and synthetic AMPs in human and experimental sepsis and their potential to provide significant improvements in the treatment of critically ill with severe

  12. Recent Advances in Peptide Immunomodulators.

    Science.gov (United States)

    Zerfas, Breanna L; Gao, Jianmin

    2015-01-01

    With the continued rise in antibiotic-resistant bacteria, there is an immense need for the development of new therapeutic agents. Host-defense peptides (HDPs) offer a unique alternative to many of the current approved antibiotics. By targeting the host rather than the pathogen, HDPs offer several benefits over traditional small molecule drug treatments, such as a slower propensity towards resistance, broad-spectrum activity and lower risk of patients developing sepsis. However, natural peptide structures have many disadvantages as well, including susceptibility to proteolytic degradation, significant costs of synthesis and host toxicity. For this reason, much work has been done to examine peptidomimetic structures, in the hopes of finding a structure with all of the desired qualities of an antibiotic drug. Recently, this research has included synthetic constructs that mimic the behavior of HDPs but have no structural similarity to peptides. This review article focuses on the progression of this field of research, beginning with an analysis of a few prominent examples of natural HDPs and moving on to describe how the information learned by studying them have led to the current design platforms.

  13. A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

    Science.gov (United States)

    Stan-Lotter, Helga; Hochstein, Lawrence I.

    1989-01-01

    A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase.

  14. Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance.

    Science.gov (United States)

    Gasic, Ksenija; Korban, Schuyler S

    2007-07-01

    Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.

  15. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells

    Directory of Open Access Journals (Sweden)

    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS. We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  16. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells.

    Science.gov (United States)

    Slomiany, Bronislaw L; Slomiany, Amalia

    2010-01-01

    Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS). We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  17. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Science.gov (United States)

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata.

  18. Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

    Science.gov (United States)

    Maruyama, T; Ito, M; Honda, G

    2001-10-01

    We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

  19. Characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene from Ginkgo biloba.

    Science.gov (United States)

    Kim, Sang-Min; Kim, Soo-Un

    2010-02-01

    Diterpene trilactone ginkgolides, one of the major constituents of Ginkgo biloba extract, have shown interesting bioactivities including platelet-activating factor antagonistic activity. 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), converting 2-C-methyl-d-erythritol-2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate, is the penultimate enzyme of the seven-step 2-C-methyl-d-erythritol 4-phosphate pathway that supplies building blocks for plant isoprenoids of plastid origin such as ginkgolides and carotenoids. Here, we report on the isolation and characterization of the full-length cDNA encoding HDS (GbHDS, GenBank accession number: DQ251630) from G. biloba. Full-length cDNA of GbHDS, 2,763 bp long, contained an ORF of 2,226 bp encoding a protein composed of 741 amino acids. The theoretical molecular weight and pI of the deduced mature GbHDS of 679 amino acid residues are 75.6 kDa and 5.5, respectively. From 2 weeks after initiation of the culture onward, transcription level of this gene in the ginkgo embryo roots increased to about two times higher than that in the leaves. GbHDS was predicted to possess chloroplast transit peptide of 62 amino acid residues, suggesting its putative localization in the plastids. The transient gene expression in Arabidopsis protoplasts confirmed that the transit peptide was capable of delivering the GbHDS protein from the cytosol into the chloroplasts. The isolation and characterization of GbHDS gene enabled us to further understand the role of GbHDS in the terpenoid biosynthesis in G. biloba.

  20. Identification of novel human immunodeficiency virus type 1-inhibitory peptides based on the antimicrobial peptide database.

    Science.gov (United States)

    Wang, Guangshun; Watson, Karen M; Peterkofsky, Alan; Buckheit, Robert W

    2010-03-01

    To identify novel anti-HIV-1 peptides based on the antimicrobial peptide database (APD; http://aps.unmc.edu/AP/main.php), we have screened 30 candidates and found 11 peptides with 50% effective concentrations (EC(50)) of 1, increases in the Arg contents of amphibian maximin H5 and dermaseptin S9 peptides and the database-derived GLK-19 peptide improved the TIs. These examples demonstrate that the APD is a rich resource and a useful tool for developing novel HIV-1-inhibitory peptides.