title: Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase.
abstract: Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique desosamine-anchoring functionality via a heretofore under-explored "substrate engineering" strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the desosamine glycoside via an acetal linkage (referred to as "carbolides") in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.
authors.affiliation: Department of Medicinal Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
authors.forename: Mani Raj
authors.initials: MR
authors.lastname: Chaulagain
authors.forename: Allison R
authors.initials: AR
authors.lastname: Knauff
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Podust
authors.forename: John
authors.initials: J
authors.lastname: Montgomery
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000606:Amino Sugars / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D002853:Chromatography, Liquid; D003516:Cycloparaffins / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D057075:Enzyme Assays; D006900:Hydroxylation / Q000187:drug effects; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction / Q000187:drug effects; D015202:Protein Engineering; D055672:Static Electricity; D013237:Stereoisomerism; D013302:Streptomyces / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D000606:Amino Sugars; D000900:Anti-Bacterial Agents; D003516:Cycloparaffins; D018942:Macrolides; C110125:desosamine; D003577:Cytochrome P-450 Enzyme System; C442772:PikC cytochrome P450 hydroxylase