Department of Chemistry, Portland State University, Portland, OR 97207, USA.
J Biol Chem. 2012 Aug 31;287(36):30560-70. doi: 10.1074/jbc.M112.388538. Epub 2012 Jul 11.
The enzyme QueF catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ(0)) to 7-aminomethyl-7-deazaguanine (preQ(1)), the only nitrile reduction reaction known in biology. We describe here two crystal structures of Bacillus subtilis QueF, one of the wild-type enzyme in complex with the substrate preQ(0), trapped as a covalent thioimide, a putative intermediate in the reaction, and the second of the C55A mutant in complex with the substrate preQ(0) bound noncovalently. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces. In both structures, a preQ(0) molecule is bound at eight sites, and in the wild-type enzyme, it forms a thioimide covalent linkage to the catalytic residue Cys-55. Both structural and transient kinetic data show that preQ(0) binding, not thioimide formation, induces a large conformational change in and closure of the active site. Based on these data, we propose a mechanism for the activation of the Cys-55 nucleophile and subsequent hydride transfer.
酶 QueF 催化腈基的还原 7-氰基-7-脱氮鸟嘌呤（前 Q(0)）到 7-氨基甲基-7-脱氮鸟嘌呤（前 Q(1)），这是生物学中唯一已知的腈还原反应。我们在这里描述了枯草芽孢杆菌 QueF 的两个晶体结构，一个是野生型酶与底物前 Q(0)形成的复合物，其中前 Q(0)被捕获为硫代亚胺，这是反应的一个假定中间体，另一个是 C55A 突变体与非共价结合的底物前 Q(0)形成的复合物。QueF 酶形成一个不对称的隧道折叠同源二十聚体，由两个头对头的五聚体亚基组成，在亚基界面上有 10 个活性位点。在这两种结构中，前 Q(0)分子结合在八个位点上，在野生型酶中，它与催化残基 Cys-55 形成硫代亚胺共价键。结构和瞬态动力学数据都表明，前 Q(0)的结合而不是硫代亚胺的形成诱导了活性位点的大构象变化和关闭。基于这些数据，我们提出了一种激活 Cys-55 亲核试剂和随后进行氢转移的机制。
