Abstract 1465 Mechanistic studies of the radical SAM enzyme OxsB in oxentanocin A biosynthesis

Study Objective: Radical S-adenosyl-l-methionine (SAM) enzymes are currently the largest known group of metalloenzymes and catalyze an extraordinarily broad range of chemical reactions via catalytic cycles involving free radical intermediates. All radical SAM enzymes are unified by the use of an active site [Fe4S4] cluster that can reductively cleave SAM and thus initiate the radical-mediated catalytic cycle. A subgroup of radical SAM enzymes also bind cobalamin, which is typically utilized to methylate substrate molecules at otherwise unactivated centers. However, OxsB is a cobalamin-dependent radical SAM enzyme that participates in the biosynthetic conversion of 2'-deoxyadenosine 5'-monophosphate (2dAMP) to yield the antiviral and antibiotic agent oxetanocin A, which contains a four-membered oxetane ring. Unlike most other cobalamin-dependent radical SAM enzymes, OxsB operates together with the phosphohydrolase OxsA to catalyze oxidative ring contraction of the furanose ring in the substrate to yield the oxetane. The principal research objectives are thus to understand how OxsB and OxsA work together to biosynthetically generate the four-membered ring in oxetanocin A and elucidate the cobalamin-dependent radical SAM chemistry that facilitates oxidative ring contraction. Statement of Methods: A multidisciplinary approach is utilized to address this challenging biochemical system. Molecular genetics is used to prepare and isolate enzymes for in vitro study. Organic synthesis is leveraged to prepare substrates for screening as well as derivatives and isotopologs for mechanistic studies via in vitro assays. Cutting edge spectroscopic methods are used to characterize isolated products. X-ray crystallography is employed to study the active site structure of the enzymes in the presence of bound substrates and cofactors. Summary of Results: Current results demonstrate that OxsB and OxsA work sequentially to construct the oxetane ring in oxetanocin A. Recent findings indicate that OxsB catalyzes an oxidative intramolecular C‒C bond formation in 2dAMP to yield a highly strained [2.1.0]-bicyclic intermediate. This unstable intermediate undergoes ready nonenzymatic decomposition in the absence of OxsA to yield an isomer of oxetanocin aldehyde phosphate. In the presence of OxsA, however, a regioselective C‒C bond cleavage is catalyzed to afford the biosynthetic oxetanocin A aldehyde phosphate product. Preliminary mechanistic work suggests that the cobalamin cofactor may facilitate C‒C bond formation by stabilizing a radical intermediate. These results will be discussed in the general context of understanding the chemistry of cobalamin-dependent radical SAM enzymes as well as the biosynthesis of oxetane nucleosides. National Institutes of Health: GM035906, GM040541 Welch Foundation: F-1511