Carboxyl-terminal and arg38 are essential for activity of the 7α-hydroxysteroid dehydrogenase from clostridium absonum

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 (kcat/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 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. © 2014 bentham science publishers.