Bacterial oxidation of steroids. II. Studies on the enzymatic mechanism of ring A dehydrogenation.

This paper describes the properties and partial purification from Pseudomonas testosteroni of enzymes concerned with the introduction of unsaturation into ring A of 3-ketosteroids. These enzymes promote the formation of the A1e4-diene-3-one structure from steroids bearing C-19 angular methyl groups and the formation of ring A phenols from lQ-nor-steroids. P. testosteroni is a strict aerobe which can satisfy its requirement for organic carbon compounds exclusively with certain steroids (1, 2). This microorganism interconverts a number of hydroxyand ketosteroids by adaptive (steroid-induced) pyridine nucleotide-linked hydroxysteroid dehydrogenases (3,4). Subsequent steps in the oxidative degradation of steroids are dehydrogenations of ring A which lead to the formation of 1,4-androstadiene-3,17-dione from androstane-3,17-dione, 1-androstene-3,17-dione, testosterone, 4-androstene-3,17-dione or etiocholan-17@-ol-3-one. Under similar conditions 19-nor-testosterone, 4-estrene-a, l’l-dione or 1-estrene-3,17-dione are converted to estrone. These transformations by intact bacterial cells are described separately (2). Evidence will be presented that probably three distinct enzymes are involved in these dehydrogenation reactions, namely, a Ai-dehydrogenase and two A4-dehydrogenases, acting specifically on steroids in which the A :B ring fusion is trans (Aa-5~ dehydrogenase) and cis (A4-5&dehydrogenase), respective1y.l The Aland A4-5a-dehydrogenases have been partially purified and some of their properties examined. These enzymes apparently catalyze a direct removal of hydrogen from adjacent carbon