Tanabe T, Tanaka N, Uchikawa K, Kabashima T, Ito K, Nonaka T, Mitsui Y, Tsuru M, Yoshimoto T
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki, 852-8521, Japan.
J Biochem. 1998 Sep;124(3):634-41. doi: 10.1093/oxfordjournals.jbchem.a022159.
The Escherichia coli 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH; EC 1.1.1.159) has been the subject of our studies, including the cloning of its gene, and determination of the crystal structures of its binary and ternary complexes [J. Bacteriol. 173, 2173-2179 (1991); Biochemistry 35, 7715-7730 (1996)]. Through these studies, the Ser146, Tyr159, and Lys163 residues were found to be involved in its catalytic action. In order to clarify the roles of these residues, we constructed six single mutants of 7alpha-HSDH, Tyr159-Phe (Y159F), Tyr159-His (Y159H), Lys163-Arg (K163R), Lys163-Ile (K163I), Ser146-Ala (S146A), and Ser146-His (S146H), by site-directed mutagenesis. These mutants were overexpressed in E. coli WSD, which is a 7alpha-HSDH null strain, and the expressed enzymes were purified to homogeneity. The kinetic constants of the mutant enzymes were determined, and the structures of the Y159F, Y159H, and K163R mutants were analyzed by X-ray crystallography. The Y159F mutant showed no activity, while the Y159H mutant exhibited 13.3% of the wild-type enzyme activity. No remarkable conformational change between the Y159F (or Y159H) and wild-type proteins was detected on X-ray crystallography. On the other hand, the K163I mutant showed just 5.3% of the native enzyme activity, with a 8. 5-fold higher Kd. However, the K163R mutant retained 64% activity, and no remarkable conformational change was detected on X-ray crystallography. In the cases of the S146A and S146H mutants, the activities fairly decreased, with 20.3 and 35.6% of kcat of the wild-type, respectively. The data presented in this paper confirm that Tyr159 acts as a basic catalyst, that Lys163 binds to NAD(H) and lowers the pKa value of Tyr159, and that Ser146 stabilizes the substrate, reaction intermediate and product in catalysis.
大肠杆菌7α-羟基类固醇脱氢酶（7α-HSDH；EC 1.1.1.159）一直是我们的研究对象，包括其基因的克隆以及其二元复合物和三元复合物晶体结构的测定[《细菌学杂志》173, 2173 - 2179 (1991)；《生物化学》35, 7715 - 7730 (1996)]。通过这些研究，发现Ser146、Tyr159和Lys163残基参与其催化作用。为了阐明这些残基的作用，我们通过定点诱变构建了7α-HSDH的六个单突变体，即Tyr159-Phe（Y159F）、Tyr159-His（Y159H）、Lys163-Arg（K163R）、Lys163-Ile（K163I）、Ser146-Ala（S146A）和Ser146-His（S146H）。这些突变体在7α-HSDH基因缺失菌株大肠杆菌WSD中过表达，表达的酶被纯化至同质。测定了突变酶的动力学常数，并通过X射线晶体学分析了Y159F、Y159H和K163R突变体的结构。Y159F突变体无活性，而Y159H突变体表现出野生型酶活性的13.3%。在X射线晶体学分析中，未检测到Y159F（或Y159H）与野生型蛋白之间有明显的构象变化。另一方面，K163I突变体仅表现出天然酶活性的5.3%，其解离常数（Kd）高8.5倍。然而，K163R突变体保留了64%的活性，在X射线晶体学分析中未检测到明显的构象变化。在S146A和S146H突变体的情况下，活性显著降低，分别为野生型催化常数（kcat）的20.3%和35.6%。本文给出的数据证实，Tyr159作为碱性催化剂，Lys163与NAD(H)结合并降低Tyr159的pKa值，Ser146在催化过程中稳定底物、反应中间体和产物。
