Mutagenesis of a modular polyketide synthase enoylreductase domain reveals insights into catalysis and stereospecificity.
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.
Kwan
David H
DH
Department of Biochemistry, University of Cambridge, United Kingdom. dkwan@chem.ubc.ca
Leadlay
Peter F
PF
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007783:Lactones / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016930:Saccharopolyspora / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
D007783:Lactones; D011994:Recombinant Proteins; D048630:Polyketide Synthases