Department of Biochemistry, Center in Molecular Toxicology, and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232-0146, United States.
Biochemistry. 2011 Feb 22;50(7):1274-81. doi: 10.1021/bi101861a. Epub 2011 Jan 24.
The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.07 ± 0.03 mM) and one weak (K(d2) = 1.3 ± 0.2 mM) binding site for GSH. YghU exhibits little or no GSH transferase activity with most typical electrophilic substrates but does possess a modest catalytic activity toward several organic hydroperoxides. Most notably, the enzyme also exhibits disulfide-bond reductase activity toward 2-hydroxyethyl disulfide [k(cat) = 74 ± 6 s(-1), and k(cat)/K(M)(GSH) = (6.6 ± 1.3) × 10(4) M(-1) s(-1)] that is comparable to that previously determined for YfcG. A superposition of the structures of the YghU·2GSH and YfcG·GSSG complexes reveals a remarkable structural similarity of the active sites and the 2GSH and GSSG molecules in each. We conclude that the two structures represent reduced and oxidized forms of GSH-dependent disulfide-bond oxidoreductases that are distantly related to glutaredoxin 2. The structures and properties of YghU and YfcG indicate that they are members of the same, but previously unidentified, subfamily of GSH transferase homologues, which we suggest be called the nu-class GSH transferases.
大肠杆菌中谷胱甘肽转移酶同源物 YghU 的晶体结构（1.50 Å 分辨率）和生化性质表明，该蛋白不寻常之处在于每个活性部位结合两个 GSH 分子。晶体学观察结果与两相平衡结合数据一致，表明 GSH 具有一个紧密（K(d1) = 0.07 ± 0.03 mM）和一个弱（K(d2) = 1.3 ± 0.2 mM）结合位点。YghU 对大多数典型的亲电底物几乎没有或没有 GSH 转移酶活性，但对几种有机过氧化物具有适度的催化活性。最值得注意的是，该酶对 2-羟乙基二硫醚也表现出二硫键还原酶活性 [k(cat) = 74 ± 6 s(-1)，k(cat)/K(M)(GSH) = (6.6 ± 1.3) × 10(4) M(-1) s(-1)]，与之前确定的 YfcG 相当。YghU·2GSH 和 YfcG·GSSG 复合物的结构叠加表明，活性部位和每个复合物中的 2GSH 和 GSSG 分子具有惊人的结构相似性。我们得出结论，这两个结构代表了与谷胱甘肽依赖的二硫键氧化还原酶密切相关的还原和氧化形式，这些酶与谷胱甘肽还原酶 2 不同。YghU 和 YfcG 的结构和性质表明它们是相同的，但以前未被识别的谷胱甘肽转移酶同源物的亚家族成员，我们建议将其称为 nu 类谷胱甘肽转移酶。
