Biotin synthase mechanism: an overview

Biotin synthase, a member of the ‘radical SAM’ (S-adenosylmethionine) family, converts DTB (dethiobiotin) into biotin. The active form of the Escherichia coli enzyme contains two (Fe-S) centres, a (4Fe-4S) and a (2Fe-2S). The (4Fe-4S)^2+/+ mediates the electron transfer required for the reductive cleavage of SAM into methionine and a DOA^• (deoxyadenosyl radical). Two DOA^•, i.e. two SAM molecules, are consumed to activate the positions 6 and 9 of DTB. A direct transfer of isotope from the labelled substrate into DOAH (deoxyadenosine) has been observed with ²H, although not quantitatively, but not with tritium. The source of the sulphur introduced to form biotin is still under debate. We have shown that the (2Fe-2S)²⁺ cluster can be reconstituted in the apoenzyme with S²⁻ and Fe²⁺. When S²⁻ was replaced by [³⁴S²⁻], [³⁵S²⁻] or Se²⁻, biotin containing mostly the sulphur isotopes or selenium was obtained. This leads us to favour the hypothesis that the (2Fe-2S) centre is the sulphur donor, which may explain the absence of turnover of the enzyme. DTBSH (9-mercaptodethiobiotin), which already contains the sulphur atom of biotin, was shown to be an alternative substrate of biotin synthase both in vivo and with a crude extract. When this compound was tested with a well-defined in vitro system, the same turnover of one and similar reaction rates were observed for DTB and DTBSH. We postulate that the same intermediate is formed from both substrates.