Further Investigation on the Turnover of Escherichia coli Biotin Synthase with Dethiobiotin and 9-Mercaptodethiobiotin as Substrates

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• 作者：Bernadette Tse Sum Bui ; Manuela Lotierzo ; Franck Escalettes ; Dominique Florentin ; and André ; e Marquet
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：December 28, 2004
• 年：2004
• 卷：43
• 期：51
• 页码：16432 - 16441
• 全文大小：157K
• 年卷期：v.43,no.51(December 28, 2004)
• ISSN：1520-4995

文摘

Biotin synthase, a member of the "radical-SAM" family, produces biotin by inserting a sulfuratom between C-6 and C-9 of dethiobiotin. Each of the two saturated carbon atoms is activated throughhomolytic cleavage of a C-H bond by a deoxyadenosyl radical, issued from the monoelectronic reductionof S-adenosylmethionine (SAM or AdoMet). An important unexplained observation is that the enzymeproduces only 1 mol of biotin per enzyme monomer. Some possible reasons for this absence of multipleturnovers are considered here, in connection with the postulated mechanisms. There is a general agreementamong several groups that the active form of biotin synthase contains one (4Fe-4S)^2+,1+ center, whichmediates the electron transfer to AdoMet, and one (2Fe-2S)²⁺ center, which is considered the sulfur source[Ugulava, N. B., Sacanell, C. J., and Jarrett, J. T. (2001) Biochemistry 40, 8352-8358; Tse Sum Bui, B.,Benda, R., Schünemann, V., Florentin, D., Trautwein, A. X., and Marquet, A. (2003) Biochemistry 42,8791-8798; Jameson, G. N. L., Cosper, M. M., Hernandez, H. L., Johnson, M. K., and Huynh, B. H.(2004) Biochemistry 43, 2022-2031]. An alternative hypothesis considers that biotin synthase has apyridoxal phosphate (PLP)-dependent cysteine desulfurase activity, producing a persulfide which couldbe the sulfur donor. The absence of turnover was explained by the inhibition due to deoxyadenosine, anend product of the reaction [Ollagnier-de Choudens, S., Mulliez, E., and Fontecave, M. (2002) FEBSLett. 535, 465-468]. In this work, we show that our purified enzyme has no cysteine desulfurase activityand the required sulfide has to be added as Na₂S. It cannot be replaced by cysteine, and consistently, PLPhas no effect. We observed that deoxyadenosine does not inhibit the reaction either. On the other hand,if the (2Fe-2S)²⁺ center is the sulfur source, its depletion after reaction could explain the absence ofturnover. We found that after addition of fresh cofactors, including Fe²⁺ and S^2-, either to the assaywhen one turn is completed or after purification of the reacted enzyme by different techniques, only asmall amount of biotin (0.3-0.4 equiv/monomer) is further produced. This proves that an active enzymecannot be fully reconstituted after one turn. When 9-mercaptodethiobiotin, which already contains thesulfur atom of biotin, is used as the substrate, the same turnover of one is observed, with similar reactionrates. We postulate that the same intermediate involving the (2Fe-2S) cluster is formed from both substrates,with a rate-determining step following the formation of this intermediate.