Roles of the Ser146, Tyr159, and Lys163 Residues in the Catalytic Action of 7.ALPHA.-Hydroxysteroid Dehydrogenase from Escherichia coli.

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概要

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• The Escherichia coli 7α-hydroxysteroid dehydrogenase (7α-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 7α-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 7α-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.

• 社団法人 日本生化学会の論文

著者

• Kabashima Tsutomu

  School Of Pharmaceutical Sciences Nagasaki University

• Tsuru Masato

  Information Science Center Nagasaki University

• Nonaka Takamasa

  Department Of Bioengineering Nagaoka University Of Technology

• Ito Kiyoshi

  School Of Pharmaceutical Sciences Nagasaki University

• TANABE Tetsurou

  School of Pharmaceutical Sciences, Nagasaki University

• TANAKA Nobutaka

  School of Pharmaceutical Sciences, Showa University

• MITSUI Yukio

  Department of BioEngineering Nagaoka University of Technology

• Tanabe Tetsurou

  School Of Pharmaceutical Sciences Nagasaki University

• Uchikawa Kouichiro

  School Of Pharmaceutical Sciences Nagasaki University

• Yoshimoto Tadashi

  School of Pharamaceutical Sciences, Nagasaki University

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