The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4.
The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-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 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-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 alpha-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 6beta-hydroxylation.
Roussel
F
F
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1031, USA. farousse@utmb.edu
Khan
K K
KK
Halpert
J R
JR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D007664:Ketosteroids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011374:Progesterone / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D007664:Ketosteroids; D011994:Recombinant Proteins; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human