The Biochemical Basis for Stereochemical Control in Polyketide Biosynthesis

Valenzano, Chiara R.; Lawson, Rachel J.; Chen, Alice Y.; Khosla, Chaitan; Cane, David E.

doi:10.1021/ja908296m

Cited by 73 publications

(127 citation statements)

References 54 publications

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“…Recombinant DEBS KR1 reduces racemic 2-methyl-3-ketopentanoyl-SNAc exclusively to a syn-(2S,3R)-2-methyl-3-hydroxy diketide (NDK-SNAc), thereby demonstrating that this reductase is completely specific not only for reduction on the re-face of the ketone carbonyl but also for the natural L-configuration of the adjacent 2-methyl group, consistent with the overall stereospecificity of DEBS module 1 (59,60 (Fig. 4a) (61,62). DEBS KR2, KR5, and KR6 are completely diastereoselective, generating a product with the identical (2R,3S)-2-methyl-3-hydroxy stereochemistry as that of the parent DEBS modules from which they had been derived ( Figs.…”

Section: Stereochemistry Of Kr-catalyzed 3-ketoacyl-acp Reductionssupporting

confidence: 52%

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Cane

2010

Journal of Biological Chemistry

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Since the discovery and sequencing of 6-deoxyerythronolide B synthase 20 years ago, this exceptionally large, multifunctional protein remains the paradigm for the understanding of the structure and biochemical function of modular polyketide synthases. The broad-spectrum macrolide antibiotic erythromycin is one of several hundred closely related, branched chain, polyoxygenated polyketides, many of which are widely used in human and veterinary medicine as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. The multistep assembly of the parent macrocyclic aglycon, 6-deoxyerythronolide B (6-dEB), 2 is controlled by a large (2 MDa) modular protein known as 6-dEB synthase (DEBS) (1-4). The biosynthetic intermediates never leave the polyketide synthase (PKS) but are passed along the DEBS assembly line from one acyl carrier protein (ACP) domain to the next. In fact, DEBS has served as the prototype of modular PKS gene clusters, dozens of which of both known and unknown function have now been sequenced from bacterial sources (5-7).Each homodimeric DEBS subunit contains two 160 -200-kDa protein modules, each responsible for a single round of polyketide chain extension and functional group modification. Within each module are several catalytic domains of 100 -400 amino acids each that are analogous in structure, function, and organization to the corresponding fatty acid synthase (FAS) components (8 -10). All six DEBS modules contain three core domains consisting of 1) a ␤-ketoacyl-ACP synthase (the ketosynthase (KS) domain) that catalyzes the key polyketide chainbuilding reaction, a decarboxylative condensation of a methylmalonyl-ACP building block with the polyketide chain provided by the upstream PKS module (see Figs. 1 and 2); 2) an acyltransferase (AT) domain that specifically loads the methylmalonyl-CoA extender unit onto the flexible 18-Å phosphopantetheine arm of the ACP domain; and 3) the ACP domain itself, which carries the polyketide biosynthetic intermediates from domain to domain and then delivers the resulting product to the KS domain of the downstream module. Additional FASlike domains are responsible for modification of the oxidation state and stereochemistry of the growing ACP-bound intermediates: a ␤-ketoacyl-ACP reductase (KR) domain, a dehydratase (DH) domain, and an enoyl reductase (ER) domain. At the N terminus of the most upstream module is a loading didomain that primes the KS domain of module 1 with the propionyl starter unit. Finally, cyclization of the full-length macrocyclic polyketide and release of 6-dEB are controlled by a dedicated thioesterase domain located at the C terminus of the most downstream module. Programming of Polyketide BiosynthesisThe chain length, substitution pattern, and oxidation level of the initially generated, full-length heptaketide 6-dEB are the direct consequence of the number of DEBS modules as well as the domain composition of each module (Fig. 1) (8 -11). The striking colinearity between the organization of the constituent biosynthetic do...

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Section: Stereochemistry Of Kr-catalyzed 3-ketoacyl-acp Reductionssupporting

confidence: 52%

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Cane

2010

Journal of Biological Chemistry

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“…Notably, such an abortive back-transfer would be expected to result in eventual inactivation of the KS3 domain by the deadend intermediate. Similarly, the KR2 domain may be unable to process tetraketide or longer substrates, analogous to the previously observed strict specificity of the KR1 domain for only 3-ketoacyl-ACP diketide substrates (30). If these observations are general, then large-scale reprogramming of PKS assembly lines will require consideration of the intrinsic substrate tolerance of both KS and KR active sites in choosing heterologous modules that could be combined to yield unique catalytic cycles.…”

Section: Resultsmentioning

confidence: 65%

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Kapur¹,

Lowry

Yuzawa³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.A fundamental challenge to our understanding of multimodular polyketide synthases (PKSs) is the ability to explain the unidirectional translocation of growing polyketide chains through these enzymatic assembly lines. The 6-deoxyerythronolide B synthase (DEBS; Fig. 1) is arguably the most well studied example of assembly line PKSs (1-4). Each module of DEBS has an acyl carrier protein (ACP) that collaborates with the β-ketosynthase (KS) domain of the same module to catalyze a single round of polyketide chain elongation ( Fig. 2). At this point, the ACP-bound intermediate is precluded from back-transfer to the same KS domain and is instead translocated to the KS domain of the downstream module (Fig. 2).In the course of our investigations into the mechanism of intermodular chain translocation (Fig. 2, reaction 1) and intramodular chain elongation (Fig. 2, reaction 4) within DEBS, we discovered that the specificity of these two reactions is controlled by protein-protein interfaces involving distinct regions of the ACP domain (5). In the present study, we have used site-directed mutagenesis to identify ACP residues that contribute to the observed specificity. In turn, these residue-level constraints were exploited to validate the proposed structural model for ACP docking during chain elongation (5), as well as to develop an analogous in silico model for chain translocation. Generalization of these models to three naturally occurring PKSs has revealed a programming pattern that establishes a ratchet mechanism that accurately explains the unique chain translocation pathway of each PKS. As a test of this ratcheting mechanism in PKS assembly lines, we engineered a module of DEBS to iteratively catalyze two successive rounds of chain elongation instead of only one. Results and DiscussionIdentification of ACP Residues That Contribute to Chain Elongation Specificity. All ACPs from fatty acid and polyketide synthases are all-helical bundles comprised of three major α-helices connected by two structured loops (Fig. 3A). In earlier work (5) we showe...

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“…[20] Stereospecificity of the KR domains in the front of the active DH domains have been speculated by comparison with those of the other KR domains indicating that 2R-methyl and 3R-hydroxyl functionality could be formed. [21,22] Because KR10 should afford 2S-methyl and 3R-hydroxyl functionality judging from the stereochemistry of FD-891, DH10 might not accept the elongated polyketide intermediate having an S configuration of the a-substituent for dehydration, and thus the skipped substrate would be processed by the next module, module 11. The catalytic region of KR10 domain has two unique amino acid residues compared with the other B1 type KR domains (KR3, KR4, KR5, KR6, KR7, KR8, KR9, KR11, and KR12) indicating a B2-type KR domain (Supporting Information).…”

Section: Identification Of the Gene Clustermentioning

confidence: 99%

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

et al. 2010

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FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.

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The Biochemical Basis for Stereochemical Control in Polyketide Biosynthesis

Cited by 73 publications

References 54 publications

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Programming of Erythromycin Biosynthesis by a Modular Polyketide Synthase

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

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