A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis

Abstract

Summary Oxetanocin-A (OXT-A, 1) is a potent antitumor, antiviral, and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne, Bacillus megaterium gene cluster that contains four genes, oxsA, oxsB, oxrA, and oxrB. Here, we show that the oxsA and oxsB genes are both required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, revealed that OXT-A is derived from 2′-deoxyadenosine phosphate in an OxsB-catalyzed ring contraction reaction initiated by H-atom abstraction from C2′. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme for which there are an estimated 7000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl-l-methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration

“…9,11,21 This hypothesis is consistent with the only known crystal structure of a Cbl-dependent radical SAM enzyme, OxsB. 25 Though not a RSMT, the substrate binding site of OxsB is postulated to be interposed equidistant between the C5′ of SAM and the Co of the Cbl cofactor.…”

C–C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products

“…80 A structurally homologous N-terminal domain is also found in B 12 -dependent radical SAM enzymes where it binds the cobalamin cofactor. 81 While most functionally characterized members of this group so far are responsible for methylation of unactivated carbon or phosphorus centers, recent studies have revealed that some of these enzymes are responsible for carbon skeleton formations where the roles of B 12 or the N-terminal domain itself are still unclear. Using the representative members, NikJ/PolH, OxsB and BchE, we will discuss the mechanism of C–C bond formation in this family.…”

“…20a). 81 Both of these genes have been heterologously expressed and characterized. Recombinant OxsA was shown to catalyze hydrolysis of phosphorylated OXT-A compounds.…”

“…99 Recombinant OxsB harbored a single [4Fe–4S] cluster and hydroxocobalamin, consistent with its annotation as a cobalamin-dependent radical SAM enzyme. 81 Initially, no catalytic activity was observed in OxsB assays using various nucleosides and nucleotides. However, product formation was observed when OxsB assays were performed in the presence of OxsA using mono, di, or triphosphorylated 2′-deoxyadenosine as substrates.…”

“…However, product formation was observed when OxsB assays were performed in the presence of OxsA using mono, di, or triphosphorylated 2′-deoxyadenosine as substrates. 81 The product of these reactions was characterized as dehydro-OXT-A phosphate ( 26 , Fig. 20a).…”