Carboxyl-Terminal and Arg38 are Essential for Activity of the 7α-Hydroxysteroid Dehydrogenase from Clostridium absonum
Deshuai Lou, Bochu Wang, Jun Tan and Liancai Zhu
Affiliation: College of Bioengineering, Chongqing University, No.174, Shapingba Main Street, Chongqing, P.R. China.
7α-hydroxysteroid dehydrogenase (7α-HSDH, EC 126.96.36.199), 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 (kcat/Km) 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 Mg2+ (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.
Keywords: Bile acid, hydroxysteroid dehydrogenase, site-directed mutagenesis, taurochenodeoxycholic acid, tauroursodeoxycholic
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