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An Oxetane-Based Polyketide Surrogate To Probe Substrate Binding in a Polyketide Synthase.

ABSTRACT: Polyketides are a large class of bioactive natural products with a wide range of structures and functions. Polyketides are biosynthesized by large, multidomain enzyme complexes termed polyketide synthases (PKSs). One of the primary challenges when studying PKSs is the high reactivity of their poly-?-ketone substrates. This has hampered structural and mechanistic characterization of PKS-polyketide complexes, and, as a result, little is known about how PKSs position the unstable substrates for proper catalysis while displaying high levels of regio- and stereospecificity. As a first step toward a general plan to use oxetanes as carbonyl isosteres to broadly interrogate PKS chemistry, we describe the development and application of an oxetane-based PKS substrate mimic. This enabled the first structural determination of the acyl-enzyme intermediate of a ketosynthase (KS) in complex with an inert extender unit mimic. The crystal structure, in combination with molecular dynamics simulations, led to a proposed mechanism for the unique activity of DpsC, the priming ketosynthase for daunorubicin biosynthesis. The successful application of an oxetane-based polyketide mimic suggests that this novel class of probes could have wide-ranging applications to the greater biosynthetic community interested in the mechanistic enzymology of iterative PKSs.

SUBMITTER: Ellis BD

PROVIDER: S-EPMC6625825 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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An Oxetane-Based Polyketide Surrogate To Probe Substrate Binding in a Polyketide Synthase.

Ellis Bryan D BD Milligan Jacob C JC White Alexander R AR Duong Vy V Altman Pilar X PX Mohammed Lina Y LY Crump Matthew P MP Crosby John J Luo Ray R Vanderwal Christopher D CD Tsai Shiou-Chuan SC

Journal of the American Chemical Society 20180410 15

Polyketides are a large class of bioactive natural products with a wide range of structures and functions. Polyketides are biosynthesized by large, multidomain enzyme complexes termed polyketide synthases (PKSs). One of the primary challenges when studying PKSs is the high reactivity of their poly-β-ketone substrates. This has hampered structural and mechanistic characterization of PKS-polyketide complexes, and, as a result, little is known about how PKSs position the unstable substrates for pro ...[more]

PMID: 29620883

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Project description:BackgroundPolyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.ResultsThe substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.ConclusionsThese results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Dataset Information

An Oxetane-Based Polyketide Surrogate To Probe Substrate Binding in a Polyketide Synthase.

Publications

An Oxetane-Based Polyketide Surrogate To Probe Substrate Binding in a Polyketide Synthase.

Similar Datasets

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