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 (AdoMet) which is cleaved into methionine (Met) and 5'-deoxyadenosine (DOA) in equimolar amounts (Guianvarc'h, D.; Florentin, D.; Tse Sum Bui, B.; Nunzi, F.; Marquet, A. Biochem. Biophys. Res. Commun. 1997, 236, 402-406). To look at 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.; Courtieu, J. Tetrahedron Lett 1998, 39, 7499-7502) have been synthesized and incubated with biotin synthase in the presence of AdoMet. Mass spectrometry analysis revealed that deuterium was indeed transferred from the substrate into deoxyadenosine, bringing the first experimental evidence for the involvement of a deoxyadenosyl radical in the activation of the functionalized positions. The results also allow us to conclude that 2 mol of AdoMet are necessary for breaking the C-H bonds at positions 6 and 9.