3α‐Hydroxysteroid: NAD Oxidoreductase Activity in Crystalline Preparations of 20β‐Hydroxysteroid: NAD Oxidoreductase

Crystalline 20β‐hydroxysteroid: NAD oxidoreductase preparations from Streptomyces hydrogenans were shown to have 3α‐hydroxysteroid: NAD oxidoreductase activity. Compounds of both the 5α‐androstane and 5α‐pregnane series served as substrates.Among the compounds tested, no evidence was found of 3β‐hydroxysteroid: NAD oxidoreductase activity. The 3α‐hydroxysteroid: NAD oxidoreductase activity did not extend to compounds having 5β‐H, a 4,5 double bond, a 5,10 double bond, or 1,2 and 4,5 double bonds together. In the 5α‐H series, a 1,2 double bond prevented the compound from serving as a substrate.Substituents at C‐11, ‐16 and ‐17 altered the kinetic constants for the 3α‐hydroxysteroid: NAD oxidoreductase reaction, and in the case of an 11β‐hydroxy or 11‐oxo group the effects were similar to the effects reported for these groups for 20β‐hydroxysteroid: NAD oxidoreductase activity.Kinetic data were consistent with the view that two types of 3α‐hydroxysteroid: NAD oxidoreductase were present which involved different active centres, neither of which was the active centre for the 20β‐hydroxysteroid: NAD oxidoreductase activity. It is not yet established whether these enzymic activities are associated with the same or different molecules. The 3α‐hydroxysteroid: NAD oxidoreductase activities do not correspond to those of previously characterised enzymes (3α‐hydroxysteroid dehydrogenase and α‐hydroxycholanate dehydrogenase.