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Mutagenesis of a modular polyketide synthase enoylreductase domain reveals insights into catalysis and stereospecificity.
ACS Chem Biol. 2010 Sep 17; 5(9):829-38.AC

Abstract

Modular type I polyketide synthases (PKSs) such as the 6-deoxyerythronolide B synthase (DEBS) or the rapamycin synthase (RAPS) biosynthesize their polyketide products in a fashion similar to fatty acid biosynthesis. Each module of these enzymes consists of multiple catalytic domains. The constituent enoylreductase (ER) domain of a given module stereospecifically reduces an enzyme-bound 2-enoyl intermediate. In a recombinant model PKS containing an ER domain derived from module 13 of RAPS, we have previously used site-specific mutagenesis to identify a key active site residue that influences the stereochemistry of enoylreduction. In this study we have identified further residues involved in stereospecificity. We show here that several other residues, previously considered as catalytically important in the medium-chain dehydrogenase/reductase family of enzymes to which PKS ERs belong, are not essential for enoylreduction in polyketide biosynthesis. However, our results suggest that a lysine residue, also modeled to lie at the active site, might serve as a proton donor to the C-2 position during enoylreduction, as previously proposed for an analogously placed lysine in mammalian fatty acid synthase. These findings further highlight the close mechanistic link between fatty acid and polyketide synthases and provide useful guidance for future biosynthetic engineering of complex polyketide biosynthesis.

Authors+Show Affiliations

Department of Biochemistry, University of Cambridge, United Kingdom. dkwan@chem.ubc.caNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20666435

Citation

Kwan, David H., and Peter F. Leadlay. "Mutagenesis of a Modular Polyketide Synthase Enoylreductase Domain Reveals Insights Into Catalysis and Stereospecificity." ACS Chemical Biology, vol. 5, no. 9, 2010, pp. 829-38.
Kwan DH, Leadlay PF. Mutagenesis of a modular polyketide synthase enoylreductase domain reveals insights into catalysis and stereospecificity. ACS Chem Biol. 2010;5(9):829-38.
Kwan, D. H., & Leadlay, P. F. (2010). Mutagenesis of a modular polyketide synthase enoylreductase domain reveals insights into catalysis and stereospecificity. ACS Chemical Biology, 5(9), 829-38. https://doi.org/10.1021/cb100175a
Kwan DH, Leadlay PF. Mutagenesis of a Modular Polyketide Synthase Enoylreductase Domain Reveals Insights Into Catalysis and Stereospecificity. ACS Chem Biol. 2010 Sep 17;5(9):829-38. PubMed PMID: 20666435.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mutagenesis of a modular polyketide synthase enoylreductase domain reveals insights into catalysis and stereospecificity. AU - Kwan,David H, AU - Leadlay,Peter F, PY - 2010/7/30/entrez PY - 2010/7/30/pubmed PY - 2011/1/19/medline SP - 829 EP - 38 JF - ACS chemical biology JO - ACS Chem. Biol. VL - 5 IS - 9 N2 - Modular type I polyketide synthases (PKSs) such as the 6-deoxyerythronolide B synthase (DEBS) or the rapamycin synthase (RAPS) biosynthesize their polyketide products in a fashion similar to fatty acid biosynthesis. Each module of these enzymes consists of multiple catalytic domains. The constituent enoylreductase (ER) domain of a given module stereospecifically reduces an enzyme-bound 2-enoyl intermediate. In a recombinant model PKS containing an ER domain derived from module 13 of RAPS, we have previously used site-specific mutagenesis to identify a key active site residue that influences the stereochemistry of enoylreduction. In this study we have identified further residues involved in stereospecificity. We show here that several other residues, previously considered as catalytically important in the medium-chain dehydrogenase/reductase family of enzymes to which PKS ERs belong, are not essential for enoylreduction in polyketide biosynthesis. However, our results suggest that a lysine residue, also modeled to lie at the active site, might serve as a proton donor to the C-2 position during enoylreduction, as previously proposed for an analogously placed lysine in mammalian fatty acid synthase. These findings further highlight the close mechanistic link between fatty acid and polyketide synthases and provide useful guidance for future biosynthetic engineering of complex polyketide biosynthesis. SN - 1554-8937 UR - https://www.unboundmedicine.com/medline/citation/20666435/Mutagenesis_of_a_modular_polyketide_synthase_enoylreductase_domain_reveals_insights_into_catalysis_and_stereospecificity_ L2 - https://dx.doi.org/10.1021/cb100175a DB - PRIME DP - Unbound Medicine ER -