The rate‐limiting steps in radical‐mediated carbon‐sulfur bond formation catalyzed by biotin synthase (580.6)

Abstract

Biotin synthase is an S‐adenosylmethionine (AdoMet or SAM) radical enzyme that catalyzes the substitution of sulfur for hydrogen in dethiobiotin, generating two new C‐S bonds in the thiophane ring of biotin. In a reaction shared with other members of the Radical SAM superfamily, biotin synthase uses a [4Fe‐4S] cluster to catalyze the reduction of SAM to methionine and a 5'‐deoxyadenosyl radical. This highly reactive radical abstracts a hydrogen atom from the C9 position of dethiobiotin, presumably generating a dethiobiotinyl radical, that is then quenched by a sulfide from a nearby [2Fe‐2S] cluster. Following exchange methionine and 5'‐deoxyadenosine for a second equivalent of SAM, a second reaction sequence focused on the C6 position completes the formation of the thiophane ring. Kinetic analysis of pre‐steady‐state enzyme turnover indicates that formation of the monothiolated intermediate, 9‐mercaptodethiobiotin, is significantly slower than the subsequent ring closure. Using kinetic isotope effect (KIE) analysis, we demonstrate that hydrogen atom abstraction from the C9 methyl group is the overall rate‐limiting step, with a burst phase KIE of ~35 ‐ 40. During in vitro steady‐state turnover, regeneration of the active enzyme through FeS cluster assembly becomes partially rate‐limiting, resulting in a partial masking of this isotope effect to a yield a KIE of ~5. Finally, feeding studies in E. coli with labeled dethiobiotin exhibit a large KIE, suggesting that FeS cluster assembly is not rate‐limiting in the presence of the ISC iron‐sulfur cluster assembly systems. The implications of the very large non‐classical isotope effect will be discussed in terms of enzyme evolution and the potential biotechnological applications for AdoMet radical enzymes.Grant Funding Source: Supported by NSF (MCB‐1244632 to JTJ)