Carboxyl-Terminal and Arg38 are Essential for Activity of the 7α-Hydroxysteroid Dehydrogenase from Clostridium absonum

Author(s): Deshuai Lou, Bochu Wang, Jun Tan, Liancai Zhu

Journal Name: Protein & Peptide Letters

Volume 21 , Issue 9 , 2014

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


7α-hydroxysteroid dehydrogenase (7α-HSDH, EC, 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 acid, truncation.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2014
Published on: 07 May, 2014
Page: [894 - 900]
Pages: 7
DOI: 10.2174/0929866521666140507160050
Price: $65

Article Metrics

PDF: 23