Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine.
3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].
Akao
T
T
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University.
Akao
T
T
Hattori
M
M
Namba
T
T
Kobashi
K
K
D015096:3-Hydroxysteroid Dehydrogenases / Q000032:analysis / Q000302:isolation & purification*; D002855:Chromatography, Thin Layer; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D008970:Molecular Weight; D013379:Substrate Specificity
D015096:3-Hydroxysteroid Dehydrogenases; C056636:3-alpha-hydroxyglycyrrhetinate dehydrogenase