PURIFICATION, CHARACTERIZATION, AND ANALYTICAL APPLICATIONS OF A 178-HYDROXySTEROID DEHYDROGENASE FROM AN ALCALIGENES

By selecting for growth on testosterone or estradiol17B as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3a-, 3&, and 176-hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17B-hydroxysteroid dehydrogenase, easily separable from 3fl-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3a-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 178-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (V= 303 pmol/min/mg of protein; K , = 3.6 PM) and reacts almost equally well with estradiol-l7#3 (V,,, = 356 pmol/min/ mg; K , = 6.4 p~). It consists of apparently identical subunits (M, = 32,000) and exists in polymeric form under nondenaturing conditions (M, = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17@-hydroxysteroids which may be As-olefins or ring A phenols or have cis or trans A/B ring h ions . Substituents at other positions are tolerated, although the presence of a 16aor 16B-hydroxyl group blocks the oxidation of the 178hydroxyl function. 3&Hydroxysteroids (AIB ring fusion trans, but not cis, or A6-olefins) are very poor substrates. The application of this highly active, specific, and stable 170-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.