Publications

• Cloning and characterization of the NAD-dependent 7alpha-Hydroxysteroid dehydrogenase from Bacteroides fragilis.

  Bennett M.J., McKnight S.L., Coleman J.P.

  The NAD-linked 7alpha-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was characterized and its gene cloned. The enzyme displayed optimal activities at pH 8.5 (NAD reduction) and 6.5 (NADH oxidation). The lowest K(m) and highest V(max) values were observed with chenodeox ... >> More

  The NAD-linked 7alpha-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was characterized and its gene cloned. The enzyme displayed optimal activities at pH 8.5 (NAD reduction) and 6.5 (NADH oxidation). The lowest K(m) and highest V(max) values were observed with chenodeoxycholic acid and its conjugates. The protein had subunits of 27.4 kDa and a native size of 110 kDa, suggesting a homotetrameric composition. The enzyme was relatively thermostable, retaining 95% of initial activity after 1 h at 65 degrees C. A DNA probe based on the N-terminal amino acid sequence hybridized to a 2373-bp HindIII fragment of B. fragilis DNA. This fragment was cloned into E. coli and sequenced, revealing a 780-bp open reading frame. The predicted amino acid sequence of the ORF showed strong sequence similarity to three other bacterial 7-HSDHs, all in the short-chain dehydrogenase family. The regulation of expression of this gene is currently under investigation. << Less

  Curr. Microbiol. 47:475-484(2003) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli.

  Tanaka N., Nonaka T., Tanabe T., Yoshimoto T., Tsuru D., Mitsui Y.

  7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family. It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids. The crystal structur ... >> More

  7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family. It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids. The crystal structure of the binary (complexed with NAD+) complex of 7 alpha-HSDH has been solved at 2.3 A resolution by the multiple isomorphous replacement method. The structure of the ternary complex [the enzyme complexed with NADH, 7-oxoglycochenodeoxycholic acid (as a reaction product), and possibly partially glycochenodeoxycholic acid (as a substrate)] has been determined by a difference Fourier method at 1.8 A resolution. The enzyme 7 alpha-HSDH is an alpha/beta doubly wound protein having a Rossmann-fold domain for NAD (H) binding. Upon substrate binding, large conformation changes occur at the substrate binding loop (between the beta F strand and alpha G helix) and the C-terminal segment (residues 250-255). The variable amino acid sequences of the substrate-binding loop appear to be responsible for the wide variety of substrate specificities observed among the enzymes of the SDR family. The crystal structure of the ternary complex of 7 alpha-HSDH, which is the only structure available as the ternary complex among the enzymes of the SDR family, indicates that the highly conserved Tyr159 and Ser146 residues most probably directly interact with the hydroxyl group of the substrates although this observation cannot be definite due to an insufficiently characterized nature of the ternary complex. The strictly conserved Lys163 is hydrogen-bonded to both the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of NAD(H). We propose a new catalytic mechanism possibly common to all the enzymes belonging to the SDR family in which a tyrosine residue (Tyr159) acts as a catalytic base and a serine residue (Ser146) plays a subsidiary role of stabilizing substrate binding. << Less

  Biochemistry 35:7715-7730(1996) [PubMed] [EuropePMC]

• Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli.

  Tanabe T., Tanaka N., Uchikawa K., Kabashima T., Ito K., Nonaka T., Mitsui Y., Tsuru M., Yoshimoto T.

  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, 7715-7 ... >> More

  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. << Less

  J. Biochem. 124:634-641(1998) [PubMed] [EuropePMC]