title: Carboxyl-terminal and Arg38 are essential for activity of the 7α-hydroxysteroid dehydrogenase from Clostridium absonum.
abstract: 7α-hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159), one of the short-chain dehydrogenase/reductase superfamily, catalyzes the dehydrogenation of C7 hydroxyl group of the steroid skeleton of bile acids. Clostridium absonum 7α-HSDH (Ca 7α-HSDH) was cloned and heterologously expressed in Escherichia coli. The function of carboxyterminal (C-terminal) and Arg38 of Ca 7α-HSDH was investigated through truncations and site-directed mutagenesis, respectively. When 2 and 6 amino acids of C-terminal were removed, the catalytic efficiency (k(cat)/K(m)) of Ca 7α-HSDH remained 19.1% and 2.5%, respectively. Furthermore, the activity could not be detected after 8, 14 and 17 amino acids were deleted. No activity could be detected with coenzyme either NADP(+) or NAD(+) after replacement of arginine at position 38 by aspartic acid. The metal ions Mg(2+) (50 mM), Na(+) (200 mM) and K(+) (500 mM) could maximally improve the activity of Ca 7α-HSDH by 61.4%, 64.7% and 105.7%, respectively. The activity had no significant change after incubation at 4 or 25 °C for 108 h, but decreased dramatically at 37 °C. Our study confirmed that C-terminal and Arg38 were essential for the catalytic function of Ca 7α-HSDH and the enzyme activity can be improved by metal ions.
authors.forename: Deshuai
authors.initials: D
authors.lastname: Lou
authors.forename: Bochu
authors.initials: B
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Tan
authors.affiliation: College of Bioengineering, Chongqing University, No.174, Shapingba Main Street, Chongqing, P.R. China. wangbc2000@126.com.
authors.forename: Liancai
authors.initials: L
authors.lastname: Zhu
mesh_terms: D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007477:Ions / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
chemical_list: D007477:Ions; D008670:Metals; D001120:Arginine; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase