首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Tang Y  Lee TS  Kobayashi S  Khosla C 《Biochemistry》2003,42(21):6588-6595
Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.  相似文献   

2.
Li Q  Khosla C  Puglisi JD  Liu CW 《Biochemistry》2003,42(16):4648-4657
During polyketide biosynthesis, acyl carrier proteins (ACPs) perform the central role of transferring polyketide intermediates between active sites of polyketide synthase. The 4'-phosphopantetheine prosthetic group of a holo-ACP is a long and flexible arm that can reach into different active sites and provide a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. We have determined the solution structure and characterized backbone dynamics of the holo form of the frenolicin acyl carrier protein (fren holo-ACP) by nuclear magnetic resonance (NMR). Unambiguous assignments were made for 433 hydrogen atoms, 333 carbon atoms, and 84 nitrogen atoms, representing a total of 94.6% of the assignable atoms in this protein. From 879 meaningful NOEs and 45 angle constraints, a family of 24 structures has been calculated. The solution structure is composed of three major alpha-helices packed in a bundle with three additional short helices in intervening loops; one of the short helices slowly exchanges between two conformations. Superposition of the major helical regions on the mean structure yields average atomic rmsd values of 0.49 +/- 0.09 and 0.91 +/- 0.08 A for backbone and non-hydrogen atoms, respectively. Although the three-helix bundle fold is conserved among acyl carrier proteins involved in fatty acid synthases and polyketide synthases, a detailed comparison revealed that ACPs from polyketide biosynthetic pathways are more related to each other in tertiary fold than to their homologues from fatty acid biosynthetic pathways. Comparison of the free form of ACPs (NMR structures of fren ACP and the Bacillus subtilis ACP) with the substrate-bound form of ACP (crystal structure of butyryl-ACP from Escherichia coli) suggests that conformational exchange plays a role in substrate binding.  相似文献   

3.
The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of l-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.  相似文献   

4.
Type II polyketide synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific polyketide product. As part of our ongoing studies into the mechanisms and control of polyketide biosynthesis, we report the second structure of a polyketide synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3-80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the addition of a 13-residue flexible C-terminus. A comparison of the global folds of all structurally characterized ACPs is described, illustrating that PKS ACPs show clear differences as well as similarities to FAS ACPs. (15)N relaxation experiments for the protein backbone also reveal that the long loop between helices I and II is flexible and helix II, a proposed site of protein-protein interactions, shows conformational exchange. The helices of the ACP form a rigid scaffold for the protein, but these are interspersed with an unusual proportion of flexible linker regions.  相似文献   

5.
Ligon J  Hill S  Beck J  Zirkle R  Molnár I  Zawodny J  Money S  Schupp T 《Gene》2002,285(1-2):257-267
A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.  相似文献   

6.
Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2.  相似文献   

7.
E S Meadows  C Khosla 《Biochemistry》2001,40(49):14855-14861
Biosynthesis of the carbon chain backbone of the R1128 substances is believed to involve the activity of a ketosynthase/chain length factor (ZhuB/ZhuA), an additional ketosynthase (ZhuH), an acyl transferase (ZhuC), and two acyl carrier proteins (ACPs; ZhuG and ZhuN). A subset of these proteins initiate chain synthesis via decarboxylative condensation between an acetyl-, propionyl-, isobutyryl-, or butyryl-CoA derived primer unit and a malonyl-CoA derived extender unit to yield an acetoacetyl-, beta-ketopentanoyl-, 3-oxo-4-methylpentanoyl-, or beta-ketohexanoyl-ACP product, respectively. To investigate the precise roles of ZhuH, ZhuC, ZhuG, and ZhuN, each protein was expressed in Escherichia coli and purified to homogeneity. Although earlier reports had proposed that ZhuC and its homologues played a role in primer unit selection, direct in vitro analysis of ZhuC showed that it was in fact a malonyl-CoA:ACP malonyltransferase (MAT). The enzyme could catalyze malonyl transfer but not acetyl- or propionyl-transfer onto R1128 ACPs or onto ACPs from other biosynthetic pathways, suggesting that ZhuC has broad substrate specificity with respect to the holo-ACP substrate but is specific for malonyl-CoA. Thus, ZhuC supplies extender units to both the initiating and elongating ketosynthases from this pathway. To interrogate the primer unit specificity of ZhuH, the kinetics of beta-ketoacyl-ACP formation in the presence of various acyl-CoAs and malonyl-ZhuG were measured. Propionyl-CoA and isobutyryl-CoA were the two most preferred substrates of ZhuH, although acetyl-CoA and butyryl-CoA could also be accepted and elongated. This specificity is not only consistent with earlier reports demonstrating that R1128B and R1128C are the major products of the R1128 pathway in vivo, but is also in good agreement with the properties of the ZhuH substrate binding pocket, as deduced from a recently solved crystal structure of the enzyme. Finally, to investigate the molecular logic for the occurrence of not one but two ACP genes within the R1128 gene cluster, the inhibition of ZhuH-catalyzed formation of beta-ketopentanoyl-ACP was quantified in the presence of apo-ZhuG or apo-ZhuN. Both apo-proteins were comparable inhibitors of the ZhuH catalyzed reaction, suggesting that the corresponding apo-proteins can be used interchangeably during chain initiation. Together, these results provide direct biochemical insights into the mechanism of chain initiation of an unusual bacterial aromatic PKS.  相似文献   

8.
Jiralerspong S  Rangaswamy V  Bender CL  Parry RJ 《Gene》2001,270(1-2):191-200
Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae. The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins. Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly. The two β-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF. Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension. Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension. The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s. Sequence motifs have previously been identified that correlate with AT substrates. The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs. The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs. Three ACP domains occur in the modular proteins of the COR PKS. The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32–36% similarity to the two module-associated ACPs of the COR PKS. It exhibited a higher degree of similarity to the module-associated ACPs of RAPS. The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS. Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other. The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II). Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA. The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS). An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs. Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs. Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium.  相似文献   

9.
Here, the term “module” is redefined for trans‐acyltransferase (trans‐AT) assembly lines to agree with how its domains cooperate and evolutionarily co‐migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans‐AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co‐evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not—in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering.  相似文献   

10.
Mupirocin is a polyketide antibiotic produced by Pseudomonas fluorescens. The biosynthetic cluster encodes 6 type I polyketide synthase multifunctional proteins and 29 single function proteins. The biosynthetic pathway belongs to the trans-AT group in which acyltransferase activity is provided by a separate polypeptide rather than in-cis as found in the original type I polyketide synthases. Special features of this group are in-cis methyltransferase domains and a trans-acting HMG-CoA synthase-cassette which insert α- and β- methyl groups respectively while enoyl reductase domains are absent from the condensing modules. In addition, for the mupirocin system, there is no obvious loading mechanism for initiation of the polyketide chain and many aspects of the pathway remain to be elucidated. Mupirocin inhibits isoleucyl-tRNA synthetase and has been used since 1985 to help prevent infection by methicillin-resistant Staphylococcus aureus, particularly within hospitals. Resistance to mupirocin was first detected in 1987 and high-level resistance in S. aureus is due to a plasmid-encoded second isoleucyl-tRNA synthetase, a more eukaryotic-like enzyme. Recent analysis of the biosynthetic pathway for thiomarinols from marine bacteria opens up possibilities to modify mupirocin so as to overcome this resistance.  相似文献   

11.
Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.  相似文献   

12.
Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.  相似文献   

13.
We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.  相似文献   

14.
Fang J  Zhang Y  Huang L  Jia X  Zhang Q  Zhang X  Tang G  Liu W 《Journal of bacteriology》2008,190(17):6014-6025
Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of D-tetronitrose, L-amicetose, and L-digitoxose may begin with D-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to Delta chlD3 and Delta chlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.  相似文献   

15.
The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum. Similar to taxol, they are of current clinical interest as anticancer agents. Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis. Given this information, it has been possible to test the predicted functions of several modules to date. EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor. Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates. These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit. Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.  相似文献   

16.
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   

17.
The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.  相似文献   

18.
Florova G  Kazanina G  Reynolds KA 《Biochemistry》2002,41(33):10462-10471
Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens. Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH. In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis. We have shown that sgFabH activity is present in S. glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo. Isotope incorporation studies with [CD3]acetate and [13CD3]acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C. These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process. Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S. glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E. coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S. glaucescens PKS ACP). In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM). This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes. Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols. The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes. In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.  相似文献   

19.
Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (~9 kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.  相似文献   

20.
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   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号