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Oxetanocin A (OXT-A) is a potent antitumour, 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 both the oxsA and oxsB genes are 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, reveals that OXT-A is derived from a 2' -deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen 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, a family of enzymes with an estimated 7,000 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.
OXT-A (1) is a nucleoside analogue produced by Bacillus megaterium NK84-01281. The phosphorylated forms of oXt-A inhibit cellular and viral DNA polymerases2 and have shown activity against hepatitis B3 and herpes simplex viruses1, among others. The plasmid-borne gene cluster for OXT-A biosynthesis and resistance is located within the BglII-D fragment4 and contains four open reading frames that encode two HD-domain phosphohydrolases (oxsA and oxrB), a Cbldependent AdoMet radical enzyme (oxsB), and a pentapeptide repeat protein (oxrA). The simplicity of the cluster suggests that OXT-A may be produced through the rearrangement of a purine nucleoside co-opted from a primary metabolic pathway. Since AdoMet radical enzymes catalyse some of the most challenging chemical transformations, OxsB is a likely candidate for catalysing formation of the eponymous oxetane ring.
AdoMet radical enzymes contain a [4Fe-4S] cluster that, when reduced, cleaves AdoMet and produces a highly reactive S'-deoxyadenosyl radical (S'-dAdo·)5. Cbl-binding proteins, on the other hand, generate S'-dAdo· through homolytic Co-C bond cleavage of adenosylcobalmin6 and methylate nucleophilic substrates through heterolytic Co-C bond cleavage of methylcobalamin (MeCbl)6. With over 7,000 AdoMet radical enzymes now annotated as Cbl-dependent, these dual-cofactor enzymes are emerging as a new superfamily7-16. Characterized Cbl-dependent AdoMet radical enzymes catalyse methylation of unactivated C- and P-centres10,12-14,17-20, but not all reactions attributed to this family involve methylation21. Here we describe the first characterization of a non-methylating Cbl-dependent...