3zk5
PikC D50N mutant bound to the 10-DML analog with the 3-(N,N-dimethylamino)ethanoate anchoring group
Structural highlights
FunctionPIKC_STRVZ Catalyzes the hydroxylation of narbomycin to give rise to pikromycin, and of 10-deoxymethymycin (YC-17) to give rise to methymycin and neomethymycin during macrolide antibiotic biosynthesis. In addition, produces low amounts of neopicromycin, novapikromycin and novamethymycin. Requires the participation of a ferredoxin and a ferredoxin reductase for the transfer of electrons from NADPH to the monooxygenase.[1] [2] [3] [4] [5] [6] Publication Abstract from PubMedHighly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis. Directing Group-Controlled Regioselectivity in an Enzymatic C-H Bond Oxygenation.,Negretti S, Narayan AR, Chiou KC, Kells PM, Stachowski JL, Hansen DA, Podust LM, Montgomery J, Sherman DH J Am Chem Soc. 2014 Mar 21. PMID:24627965[7] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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