Pyridine nucleotide complexes with Bacillus anthracis coenzyme A-disulfide reductase:: A structural analysis of dual NAD(P)H specificity
Pyridine nucleotide complexes with Bacillus anthracis coenzyme A-disulfide reductase:: A structural analysis of dual NAD(P)H specificity
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DOI：
10.1021/bi8002204
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发表时间：
2008-05-06
期刊：
影响因子：
2.9
通讯作者：
Claiborne, Al
中科院分区：
文献类型：
--
作者：
Wallen, Jamie R.;Paige, Carleitta;Claiborne, Al
我们最近报道了CoASH是炭疽芽孢杆菌中主要的低分子量硫醇[Nicely， n.i, Parsonage， D., Paige， C., Newton， G. L., Fahey， R., Leonardi， R., Jackowski， S., Mallett， T. C.和Claiborne， A.（2007）《生物化学》46,3234 - 3245]，我们现在表征了炭疽芽孢杆菌辅酶a -二硫还原酶（CoADR, BACoADR）的动力学和氧化还原特性，并在2.30埃分辨率下确定了晶体结构。虽然金黄色葡萄球菌和伯氏疏螺旋体CoADR分别对NADPH和NADH表现出强烈的偏好，但炭疽芽孢杆菌CoADR可以同样好地使用任何一种吡啶核苷酸。在各自的NAD(P) h结合基序内的序列元素正确地反映了金黄色葡萄球菌和Bo的偏好。burgdorferi coadr，但BACoADR如何与两种吡啶核苷酸相互作用留下了问题。在约2.3埃的分辨率下，NADH和NADPH复合物的结构显示，由残基Glu 180-Thr187组成的环在NAD(P)H结合时变得有序并改变构象。NADH和NADPH与这个环几乎相同的构象相互作用；然而，后一种相互作用涉及一种新的结合模式，即NADPH的2'-磷酸指向溶剂。此外，NAD(P) h还原BACoADR结构提供了Cys42-SSCoA氧化还原中心的还原形式（Cys42-SH/CoASH）的第一个视图。Cys42-SH侧链采用了一种新的构象，其中保守的Tyr367'-OH和Tyr425'-OH与黄素si面上的新生硫醇（ate）相互作用。Y367F、Y425F和Y367,425F BACoADR突变体的动力学数据表明，Tyr425‘是催化过程中的主要质子供体，Tyr367’在Tyr425'缺失的情况下起着隐式替代供体的作用。
We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N. I., Parsonage, D., Paige, C., Newton, G. L., Fahey, R. C., Leonardi, R., Jackowski, S., Mallett, T. C., and Claiborne, A. (2007) Biochemistry 46, 3234- 3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 angstrom resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 angstrom resolution reveal that a loop consisting of residues Glu 180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367,425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.