Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

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Abstract
3,17.beta.-Hydroxysteroid dehydrogenase was enriched and purified from cytosol of S. hydrogenans. After (NH4)2SO4 precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to MW of 70,200 .+-. 2500. 3 .beta.-, 17 .beta.- and 20 .beta.-Hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accept NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 .mu.M for 5 .alpha.-dihydrotestosterone, 20 .mu.M for testosterone and 68 .mu.M for epiandrosterone in the NAD+-driven reaction, 1.8 .times. 10-4 M for NAD+ and 1.9 .times. 10-4 M for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki [inhibition constant] 1.15 .times. 10-3 M). After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 .+-. 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing column (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3-5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the figure increases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.