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

The cDNA coding for the human 3 beta-hydroxy-5-ene steroid dehydrogenase/5- ene-4-ene steroid isomerase (3 beta-HSD) has been expressed in yeast. When expressed from identical vectors except for the coding sequence, the specif ic activity of the type I is lower than that of the type II enzyme. A mutan t of the human 3 beta-HSD type II lacking the putative membrane spanning do main 1 was generated by site directed mutagenesis: its apparent K-m for pre gnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme. The influence of the kinetic properties of 3 beta-HSD in the accumulation of 17 alpha-hydroxyprogesterone was probed by co-expre ssion of the bovine 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) cDN A. The metabolism of PREG was followed with time using the membrane fractio n. Kinetic properties of the 3 beta-HSD were modulated such that its activi ty was in excess, limiting or balanced with respect to the activity of the P45017 alpha and the accumulation of intermediates and products recorded. C onditions for the generation of the by-products resulting from the 17,20-Ly ase activity of the P45017a were found. The potential applications of the s ystem are discussed. (C) 2000 Elsevier Science Ltd. All rights reserved.