Lotierzo Manuela, Raux Evelyne, Tse Sum Bui Bernadette, Goasdoue Nicole, Libot Francine, Florentin Dominique, Warren Martin J, Marquet Andrée
Université Pierre et Marie Curie-Paris 6, CNRS UMR 7613, (Synthèse, Structure et Fonction de Molécules Bioactives), FR2769, Case Courrier 182, 4 place Jussieu, 75005 Paris, France.
Biochemistry. 2006 Oct 10;45(40):12274-81. doi: 10.1021/bi060662m.
Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a 4Fe-4S cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a 2Fe-2S cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable 2Fe-2S center, can additionally accommodate an air-sensitive 4Fe-4S center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)()). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)() radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.
生物素合酶是“自由基S-腺苷甲硫氨酸（SAM）”家族的一员，催化生物素生物合成途径的最后一步，即将一个硫原子插入到脱硫生物素（DTB）中。该酶的活性形式包含两个铁硫簇，一个由半胱氨酸-53、半胱氨酸-57和半胱氨酸-60配位的4Fe-4S簇以及S-腺苷甲硫氨酸（AdoMet或SAM）共底物，另一个由半胱氨酸-97、半胱氨酸-128、半胱氨酸-188和精氨酸-260配位的2Fe-2S簇。这六个保守半胱氨酸中的每一个进行单点突变都会产生无活性的变体。在这项工作中，描述了来自Y(150)NHNLD基序的其他高度保守残基的突变体。就其簇含量和特性而言，它们具有与野生型酶相似的性质。对于所有这些突变体，分离后的形式含有一个对空气稳定的2Fe-2S中心，还可以容纳一个对空气敏感的4Fe-4S中心，该中心是在厌氧条件下与Fe(2+)和S(2-)孵育产生的。它们的光谱性质与野生型相似。然而，除了表现出微弱活性的突变体H152A外，它们都是无活性的。我们表明，在生物素生成中无活性的突变体也无法裂解AdoMet并产生脱氧腺苷自由基（AdoCH(2)()）。在H152A的情况下，5'-脱氧腺苷（AdoCH(3)）与生物素的比率为5.5±0.4，远高于野生型通常观察到的2.8±0.3的值。这揭示了无效过程的更大贡献，即AdoCH(2)()自由基被来自蛋白质或系统某些成分的氢原子淬灭。因此，在这种情况下，AdoCH(2)(*)的产生与其与DTB的C-6和C-9处的氢的反应之间的偶联效率低于野生型，这可能是由于活性位点内的几何结构受到扰动。
