Properties of Crystalline 3α-Hydroxysteroid Dehydrogenase from Pseudomonas putida

Abstract

A crystalline 3α-hydroxysteroid: NAD⁺-oxidoreductase (EC 1. 1. 1. 50) which had been obtained from the cell-free extracts of Pseudonionas putida NRRL B-11064 in the presence of added polyethylene glycol, was found to be a native monomer form with a specific activity of 63.0 and a molecular weight of 45, 000. Isoelectric focusing exhibited the enzyme to be composed of two isoenzymes: one major part focusing at pH 4.75 and a minor part focusing at pH 5.10. Whereas the enzyme was changed from the monomeric form to a dimeric one with a considerable decrease in the specific activity during the course of crystallization in the absence of the added polyethylene glycol.
The enzyme showed an absolute specificity with regard to 3α-hydroxyl group besides a high requirement for cis A:B fusion of steroids. Typical substrates are cholic acid (Km=1.33×10^-5M), deoxycholic acid, chenodeoxycholic acid, 3α-hydroxy-12-keto-9, 11-cholanoic acid, and etiocholan-3α-ol-l7-one. Conjugated bile acids such as taurocholic acid and glyco-cholic acid are also rapidly oxidized. The pH optima for oxidation of cholic acid and reduction of etiocholan-3, 17-dione were 11.5 and 7.0, respectively. The enzyme could be employed for the sensitive and specific assay of bile acids.