The Importance of SRS-1 Residues in Catalytic Specificity of Human Cytochrome P450 3A4

• Published in: Archives of biochemistry and biophysics Vol. 374; no. 2; pp. 269 - 278
• Main Authors: Roussel, Fabienne, Khan, Kishore K., Halpert, James R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.02.2000
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Base Sequence; Binding Sites; Catalytic Domain; CYP3A4; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; DNA Primers; DNA, Complementary; heterologous expression; Humans; Ketosteroids - metabolism; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Progesterone - metabolism; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; site-directed mutagenesis; steroid hydroxylation; Substrate Specificity; Testosterone - metabolism; site-directed mutagenesis; steroid hydroxylation; heterologous expression; cytochrome P450; CYP3A4
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1599

The structural basis for the regioselective hydroxylation of Δ-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6β-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2β-OH rather than 6β-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S50 values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the Vmax values for 2β-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by α-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6β-hydroxylation.