Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5 alpha-reductase (isozyme-1) I: Analysis of aromatic and hydroxylated amino acid residues

Previous studies have shown that the reduced nicotinamide adenine dinucleot ide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5 alp ha-reductase isozyme-l (r5 alpha R-1) is in a highly conserved region of th e polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino a cids within the NADPH-binding domain. The r5 alpha R-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the e xpressed enzymes showed that the mutation Y179F resulted in an similar to 4 0-fold increase in the Km for NADPH versus wild-type, with only a 2-fold in crease in the Km for testosterone. The mutants Y189F and S164A showed small er increases (4 and 6-fold) in Kms for NADPH and no significant change in t he Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5 alpha R-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an similar to 5-fold decrease in Km for NADPH and a significant increase (similar to 18-fold) in the Km for testosterone . The results suggest that the -OH functionality of Y179 is involved in cof actor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor bin ding to r5 alpha R-1 and may not be required for enzyme activity. On the ot her hand, the residue F187 may be important for the binding of both NADPH a nd testosterone. (C) 1999 Published by Elsevier Science Inc. All rights res erved.