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1.
SEARCHPKS is a software for detection and analysis of polyketide synthase (PKS) domains in a polypeptide sequence. Modular polyketide synthases are unusually large multi-enzymatic multi-domain megasynthases, which are involved in the biosynthesis of pharmaceutically important natural products using an assembly-line mechanism. This program facilitates easy identification of various PKS domains and modules from a given polypeptide sequence. In addition, it also predicts the specificity of the potential acyltransferase domains for various starter and extender precursor units. SEARCHPKS is a user-friendly tool for correlating polyketide chemical structures with the organization of domains and modules in the corresponding modular polyketide synthases. This program also allows the user to extensively analyze and assess the sequence homology of various polyketide synthase domains, thus providing guidelines for carrying out domain and module swapping experiments. SEARCHPKS can also aid in identification of polyketide products made by PKS clusters found in newly sequenced genomes. The computational approach used in SEARCHPKS is based on a comprehensive analysis of various characterized clusters of modular polyketide synthases compiled in PKSDB, a database of modular polyketide synthases. SEARCHPKS can be accessed at http://www.nii.res.in/searchpks.html. 相似文献
2.
Polyketide synthase complexes: their structure and function in antibiotic biosynthesis. 总被引:7,自引:0,他引:7
J A Robinson 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1991,332(1263):107-114
This paper gives an overview of existing knowledge concerning the structure and deduced functions of polyketide synthases active in antibiotic-producing streptomycetes. Using monensin A as an example of a structurally complex polyketide metabolite, the problem of understanding how individual strains of microorganism are 'programmed' to produce a given polyketide metabolite is first outlined. The question then arises, how is the programming of polyketide assembly related to the structural organization of individual polyketide synthase complexes at the biochemical and genetic levels? Experimental results that help to illuminate these relations are described, in particular, those giving information about the structures and deduced functions of polyketide synthases involved in aromatic polyketide biosynthesis (actinorhodin, granaticin, tetracenomycin, whiE spore pigment and an act homologous region from the monensin-producing organism), as well as the macrolide polyketide synthase active in the biosynthesis of 6-deoxyerythronolide A. 相似文献
3.
4.
The structure of the ketoreductase (KR) from the first module of the erythromycin synthase with NADPH bound was solved to 1.79 A resolution. The 51 kDa domain has two subdomains, each similar to a short-chain dehydrogenase/reductase (SDR) monomer. One subdomain has a truncated Rossmann fold and serves a purely structural role stabilizing the other subdomain, which catalyzes the reduction of the beta-carbonyl of a polyketide and possibly the epimerization of an alpha-substituent. The structure enabled us to define the domain boundaries of KR, the dehydratase (DH), and the enoylreductase (ER). It also constrains the three-dimensional organization of these domains within a module, revealing that KR does not make dimeric contacts across the 2-fold axis of the module. The quaternary structure elucidates how substrates are shuttled between the active sites of polyketide synthases (PKSs), as well as related fatty acid synthases (FASs), and suggests how domains can be swapped to make hybrid synthases that produce novel polyketides. 相似文献
5.
Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases 总被引:9,自引:0,他引:9
Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides. 相似文献
6.
Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex 总被引:1,自引:0,他引:1
Katarzyna Kuczek Krzysztof Pawlik Magdalena Kotowska Marian Mordarski 《FEMS microbiology letters》1997,157(1):195-200
An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex. 相似文献
7.
Iterative highly reducing polyketide synthases from filamentous fungi are the most complex and enigmatic type of polyketide synthase discovered to date. Here we uncover an unusual degree of programming by the hypothemycin highly reducing polyketide synthase, in which a single ketoreductase domain shows stereospecificity that is controlled by substrate length. Mapping of the structural domains responsible for this feature allowed for the biosynthesis of an unnatural diastereomer of the natural product dehydrozearalenol. 相似文献
8.
Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56 总被引:3,自引:0,他引:3
Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases. 相似文献
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10.
T Lanz S Tropf F J Marner J Schr?der G Schr?der 《The Journal of biological chemistry》1991,266(15):9971-9976
Resveratrol and chalcone synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of stilbenes and flavonoids, respectively. The stepwise condensing reactions correspond to those in other polyketide and fatty-acid synthases. This predicts that the two proteins also contain cysteines that are essential for enzyme activity because they bind the substrates. We exchanged, in both enzymes, all of the 6 conserved cysteines into alanine by site-directed mutagenesis and tested the mutants after expression of the proteins in the Escherichia coli heterologous system. Only cysteine 169 was essential in both enzymes, and inhibitor studies suggest that it is the main target of cerulenin, an antibiotic reacting with the cysteine in the active center of condensing enzymes. Most of the other exchanges led to reduced activities. In two cases, the enzymes responded differently, suggesting that the cysteines at positions 135 and 195 may be involved in the different product specificity of the two enzymes. The sequences surrounding the essential cysteine 169 revealed no similarity to the active sites of condensing enzymes in other polyketide synthases and in fatty acid biosynthesis. The available data indicate that resveratrol and chalcone synthases represent a group of enzymes that evolved independently of other condensing enzymes. 相似文献
11.
Nguyen T Ishida K Jenke-Kodama H Dittmann E Gurgui C Hochmuth T Taudien S Platzer M Hertweck C Piel J 《Nature biotechnology》2008,26(2):225-233
Modular polyketide synthases (PKSs) are giant bacterial enzymes that synthesize many polyketides of therapeutic value. In contrast to PKSs that provide acyltransferase (AT) activities in cis, trans-AT PKSs lack integrated AT domains and exhibit unusual enzymatic features with poorly understood functions in polyketide assembly. This has retarded insight into the assembly of products such as mupirocin, leinamycin and bryostatin 1. We show that trans-AT PKSs evolved in a fundamentally different fashion from cis-AT systems, through horizontal recruitment and assembly of substrate-specific ketosynthase (KS) domains. The insights obtained from analysis of these KS mosaics will facilitate both the discovery of novel polyketides by genome mining, as we demonstrate for the thailandamides of Burkholderia thailandensis, and the extraction of chemical information from short trans-AT PCR products, as we show using metagenomic DNA of marine sponges. Our data also suggest new strategies for dissecting polyketide biosynthetic pathways and engineering polyketide assembly. 相似文献
12.
Wang Y Kim JA Cheong YH Joshi Y Koh YJ Hur JS 《Journal of microbiology (Seoul, Korea)》2011,49(3):473-480
The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens
produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated.
In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence
analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS),
acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains.
This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis
in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results
demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol. 相似文献
13.
真菌聚酮合酶在代谢中可催化合成多种具有重要生物学活性的次级代谢物,所以真菌聚酮合酶正逐渐成为药学、食品科学和农学等领域的研究热点。本文综述了近五年来建立的几种分离真菌聚酮合酶基因的方法。这些方法解决了真菌中聚酮合酶基因簇难以分离的问题,为改造和利用真菌聚酮合酶以及发掘真菌聚酮化合物资源提供了强有力的手段。 相似文献
14.
Abstract
Type I polyketide synthases are known to produce a wide range of medically and industrially important polyketides. The ketosynthase
(KS) domain is required for the condensation of an extender unit onto the growing polyketide chain during polyketide biosynthesis.
KSs represent a superfamily of complex biosynthetic pathway-associated enzymes found in prokaryotes, fungi, and plants. Although
themselves functionally conserved, KSs are involved in the production of a structurally diverse range of metabolites. Degenerate
oligonucleotide primers, designed for the amplification of KS domains, amplified KS domains from a range of organisms including
cyanobacterial and dinoflagellates. KS domains detected in dinoflagellate cultures appear to have been amplified from the
less than 3-μm filtrate of the nonaxenic culture. Phylogenetic analysis of sequences obtained during this study enabled the
specific identification of KS domains of hybrid or mixed polyketide synthase/peptide synthetase complexes, required for the
condensation of an extender unit onto an amino acid starter unit. The primer sets described in this study were also used for
the detection of novel KS domains directly from environmental samples. The ability to predict function based on primary molecular
structure will be critical for future discovery and rational engineering of polyketides. 相似文献
15.
Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies. 相似文献
16.
Kapur S Worthington A Tang Y Cane DE Burkart MD Khosla C 《Bioorganic & medicinal chemistry letters》2008,18(10):3034-3038
The critical role of protein-protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein-protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis. 相似文献
17.
Bacterial type I polyketide synthases (PKSs) are complex, multifunctional enzymes that synthesize structurally diverse and medicinally important natural products. Given their modular organization, the manipulation of type I PKSs holds tremendous promise for the generation of novel compounds that are not easily accessible by standard synthetic chemical approaches. In theory, hybrid polyketide synthetic pathways can be constructed through the rational recombination of catalytic domains or modules from a variety of PKS systems; however, the general success of this strategy has been elusive, largely due to a poor understanding of the interactions between catalytic domains, as well as PKS modules. Over the past several years, a fundamental knowledge of these issues, and others, has begun to emerge, offering refined strategies for the facile engineering of hybrid polyketide pathways. 相似文献
18.
Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds. 相似文献
19.
Shao L Qu XD Jia XY Zhao QF Tian ZH Wang M Tang GL Liu W 《Biochemical and biophysical research communications》2006,345(1):133-139
Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6-methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs. 相似文献
20.
The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 ?-resolution structure of the didomain comprising the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ~600-?(2) interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the two-fold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins to access each of their cognate enzymes. The isolated ER domain showed activity toward a substrate analog, enabling us to determine the contributions of its active site residues. 相似文献