Pregnenolone metabolized to 17alpha-hydroxyprogesterone in yeast: biochemical analysis of a metabolic pathway

The cDNA coding for the human 3beta-hydroxy-5-ene steroid dehydrogenase/5-ene-4-ene steroid isomerase (3beta-HSD) has been expressed in yeast. When expressed from identical vectors except for the coding sequence, the specific activity of the type I is lower than that of the type II enzyme. A mutant of the human 3beta-HSD type II lacking the putative membrane spanning domain 1 was generated by site directed mutagenesis: its apparent K(m) for pregnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme. The influence of the kinetic properties of 3beta-HSD in the accumulation of 17alpha-hydroxyprogesterone was probed by co-expression of the bovine 17alpha-hydroxylase cytochrome P450 (P45017alpha) cDNA. The metabolism of PREG was followed with time using the membrane fraction. Kinetic properties of the 3beta-HSD were modulated such that its activity was in excess, limiting or balanced with respect to the activity of the P45017alpha and the accumulation of intermediates and products recorded. Conditions for the generation of the by-products resulting from the 17,20-Lyase activity of the P45017alpha were found. The potential applications of the system are discussed.