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Substrate controlled divergence in polyketide synthase catalysis.

ABSTRACT: Biochemical characterization of polyketide synthases (PKSs) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. In these efforts, N-acetylcysteamine thioesters have typically been employed for in vitro studies of full PKS modules as well as excised domains. However, substrate engineering approaches to control the catalytic cycle of a full PKS module harboring multiple domains remain underexplored. This study examines a series of alternatively activated native hexaketide substrates on the catalytic outcome of PikAIV, the sixth and final module of the pikromycin (Pik) pathway. We demonstrate the ability to control product formation with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring. This outcome was achieved through modifying the type of hexaketide ester employed, demonstrating the utility of substrate engineering in PKS functional studies and biocatalysis.

SUBMITTER: Hansen DA

PROVIDER: S-EPMC4379966 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Substrate controlled divergence in polyketide synthase catalysis.

Hansen Douglas A DA Koch Aaron A AA Sherman David H DH

Journal of the American Chemical Society 20150312 11

Biochemical characterization of polyketide synthases (PKSs) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. In these efforts, N-acetylcysteamine thioesters have typically been employed for in vitro studies of full PKS modules as well as excised domains. However, substrate engineering approaches to control the catalytic cycle of a full PKS module harboring multiple domains remain underexplored. This study examines a ...[more]

PMID: 25730816

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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

Substrate controlled divergence in polyketide synthase catalysis.

Publications

Substrate controlled divergence in polyketide synthase catalysis.

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