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

摘要: Abstract Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5α-reductase isozyme-1 (r5αR-1) is in a highly conserved region polypeptide sequence (residues 160–190). In this study, we investigated, by site-directed mutagenesis, role hydroxylated and aromatic amino acids within NADPH-binding domain. The r5αR-1 cDNA was cloned into pCMV vector, double strand mutagenesis method used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, Y187F, which were subsequently expressed COS-1 cells. Kinetic enzymes showed mutation Y179F resulted an ∼40-fold increase Km for NADPH versus wild-type, with only 2-fold testosterone. Y189F S164A smaller increases (4 6-fold) Kms no significant change testosterone, whereas Y189S had kinetic properties similar wild-type r5αR-1. Mutants Y179S S164T both inactive enzymes, F187Y ∼5-fold decrease (∼18-fold) results suggest -OH functionality Y179 involved cofactor binding, but not essential activity enzyme, functionalities Y189 S164 play lesser roles may be required enzyme activity. On other hand, residue F187 important