Substrate-Assisted Catalysis in Polyketide Reduction Proceeds via a Phenolate Intermediate.
SimC7 is a polyketide ketoreductase involved in biosynthesis of the angucyclinone moiety of the gyrase inhibitor simocyclinone D8 (SD8). SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, catalyzes reduction of the C-7 carbonyl of the angucyclinone, and the resulting hydroxyl is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct mechanism. To investigate this possibility, we determined the structures of SimC7 alone, with NADP(+), and with NADP(+) and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad, including the active-site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active-site mutants, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate.
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Martin
M
Schäfer
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Clare E M
CEM
Stevenson
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Barrie
B
Wilkinson
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
David M
DM
Lawson
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address: mark.buttner@jic.ac.uk.
Mark J
MJ
Buttner
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D003374:Coumarins / Q000494:pharmacology; D006027:Glycosides / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D010636:Phenols / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003374:Coumarins; D006027:Glycosides; D010636:Phenols; D061065:Polyketides; C416814:simocyclinone D8; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase