Biotin synthase mechanism: Evidence for hydrogen transfer from the substrate into deoxyadenosine

Biotin synthase, the enzyme which catalyses the conversion of dethiobiotin (DTB) to biotin has an absolute requirement for S-adenosylmethionine (AdoMe t) which is cleaved into methionine (Met) and 5'-deoxyadenosine (DOA) in eq uimolar amounts (Guianvarc'h, D.; Florentin, D.; Tse Sum Bui, B.; Nunzi, F. ; Marquet, A. Biochem. Biophys. Res. Commun. 1997, 236, 402-406). To look a t an eventual [H-2] transfer from DTB into DOA, 6,9-[H-2(5)]DTB (3a), 9-[H- 2(3)]DTB (3b), 6(S)- [H-2(1)],9-[H-2(1)]DTB and 6(R)-[H-2(1)],9-[H-2(1)]DTB (3c), (3d) (Escalettes, F.; Florentin, D.; Marquet, A.; Canlet, C.; Courti eu, J. Tetrahedron Lett 1998, 39, 7499-7502) have been synthesized and incu bated with biotin synthase in the presence of AdoMet. Mass spectrometry ana lysis revealed that deuterium was indeed transferred from the substrate int o deoxyadenosine, bringing the first experimental evidence for the involvem ent of a deoxyadenosyl radical in the activation of the functionalized posi tions. The results also allow us to conclude that 2 mol of AdoMet are neces sary for breaking the C-H bonds at positions 6 and 9.