Pseudomonas testosteroni hydroxysteroid dehydrogenases

The enzyme 3a-hydroxysteroid dehydrogenase (EC 1.1.1.50), which is isolated from Pseudomonas testosteroni, catalyzes the conversion of all 3a-hydroxycholanic acids to 3-ketochoIanic acids, with the concomitant reduction of NAD to NADH. The NADH formed in the reaction is then determined spectrophotometrically at 340 nm. To ensure complete reaction, hydrazine is usually added to bind the 3-keto products (P2). The optimum conditions for enzymatic assay include a pH of 9.0 to 9.5 and reaction temperature in the range 20 to 40 C (T13). Reaction rates for individual bile acids may not be identical, but the assay is normally carried out as an endpoint determination. Alternatively, the addition of bovine serum albumin appears to overcome the problem of variable aflinity of 3a-hy-droxysteroid dehydrogenase for different bile acids if reaction rates are to be measured (S13). 

Enzymes from various sources have been used for asymmetric reductions in organic synthesis. Microorganisms are the most important sources. There are a huge number of species (mostly in soil), containing a variety of enzymes. Commercially available microbial dehydrogenases are alcohol dehydrogenases from yeast, Ther-moanaerobium brockii (TBADH), and the hydroxysteroid dehydrogenase from Pseudomonas testosteroni. 

In a number of cases, however, enzymatic reactions do not take place specifically with respect to either enantiomer. One example of this is the demethylation of the methyl ether of racemic 8-aza-D-homoestradiol (291), which forms the racemic free phenol (292) [120] (Scheme 42). Another example is the racemic 18-norsteroid (294). Only the natural enantiomer of this compound reacts with an enzyme isolated from the bacterium Pseudomonas testosteroni, 3)3,17i3-hydroxysteroid dehydrogenase, forming the optically active 17-ketone (293). At the same time, both enantiomers of compound (294) react with another enzyme from the same source,... 

Benzoylation at the 17/3-hydroxy group, selective saponification of the 3a-acetate, and oxidation at C3 converted the trans-monoacetate (191) into the oxobenzoate (190), and from this by the introduction of a A -bond and debenzoylation 18-nortestosterone (189) was obtained. As in the case of the diol (187), only one enantiomer of the diol corresponding to the monoacetate (191) reacts with the 3/5,17/5-hydroxysteroid dehydrogenase from the bacterium Pseudomonas testosteroni, forming optically active 17-ketones. At the same time, the 3a-hydroxy groups of both enantiomers of these diols are oxidized by the 3a-hydroxysteroid dehydrogenase from the same microorganism with the formation of the racemic 3-ketones [926]. 

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