Taylor Andrew M, Farrar Christine E, Jarrett Joseph T
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Biochemistry. 2008 Sep 2;47(35):9309-17. doi: 10.1021/bi801035b. Epub 2008 Aug 9.
Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5'-deoxyadenosyl radical. In our working mechanism, the 5'-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by a sulfide from a [2Fe-2S] (2+) cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5'-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation-association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In this work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equiv of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use liquid chromatography and mass spectrometry to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at approximately 10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with the defective enzyme containing the Asn153Ser mutation. The retention of 9-mercaptodethiobiotin as a tightly bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.
生物素合酶（BS）催化硫原子向脱硫生物素（DTB）的氧化加成反应，生成生物素噻吩环。该酶是一种S-腺苷甲硫氨酸（AdoMet）自由基酶，催化AdoMet的还原裂解，生成甲硫氨酸和一个瞬态5'-脱氧腺苷自由基。在我们的作用机制中，5'-脱氧腺苷自由基通过从C6或C9上夺取一个氢原子来氧化DTB，生成一个脱硫生物素基碳自由基，该自由基被来自2Fe-2S簇的硫化物淬灭。针对另一个位置的类似反应序列生成噻吩环中的第二个C-S键。由于BS活性位点仅容纳一个AdoMet和一个DTB，因此甲硫氨酸和5'-脱氧腺苷的解离以及第二个等量AdoMet的结合必然是生物素形成过程中的中间步骤。在这些解离-结合步骤中，一个离散的DTB衍生中间体必须保持与酶结合。在这项工作中，我们证实DTB向生物素的转化伴随着2当量AdoMet的还原裂解。DTB消耗与生物素形成之间的差异表明存在一个中间体，我们使用液相色谱和质谱法证明该中间体确实是9-巯基脱硫生物素，其生成量约为总酶浓度的10%。当反应在亚化学计量水平的AdoMet或含有Asn153Ser突变的缺陷酶存在下进行时，观察到的中间体数量会增加。9-巯基脱硫生物素作为紧密结合中间体的保留与涉及逐步自由基介导的从铁硫簇中提取硫化物的机制一致。
