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.

Enzyme-mediated sulfide production for the reconstitution of [2Fe-2S] clusters into apo-biotin synthase of Escherichia coli. Sulfide transfer from cysteine to biotin.

We previously showed that biotin synthase in which the (Fe-S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe-2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.