Helices F-G are important for the substrate specificities of CYP3A7.
CYP3A7 is a member of the human CYP3A family and a major form of P450 expressed in human fetal livers. Although CYP3A7 shares nearly 90% base sequence with CYP3A4, CYP3A7 shows striking functional differences in the catalytic preference for several substrates, such as dehydroepiandrosterone (DHEA) or dehydroepiandrosterone 3-sulfate (DHEA-3S). First, to clarify the reason for the differences between CYP3A7 and CYP3A4, a homology model of CYP3A7 was constructed using the CYP3A4 crystal structure. Because these two structures were similar, four kinds of chimeric enzymes were constructed to determine which sequences are important for exhibiting the characteristics of CYP3A7. The results of kinetic analysis of DHEA and DHEA-3S 16alpha-hydroxylations by CYP3A7, CYP3A4, and CYP3A chimeras suggested that the amino acid residues from Leu(210) to Glu(279) were important to express the specificity for substrates as CYP3A7. This region was on the F and G helices of the modeled CYP3A7. Furthermore, to assess which amino acid in this sequence is important for the substrate specificity of CYP3A7, a one-point mutation of CYP3A7 to CYP3A4 was made by site-directed mutagenesis. The mutants of K224T and K244E had lost DHEA and DHEA-3S 16alpha-hydroxylation activities. The mutants also greatly decreased the metabolism of testosterone, erythromycin, nevirapine, and triazolam relative to those activities of CYP3A7 wild-type enzyme. From these results, it is expected that CYP3A7 can recognize specific substrates using the lysines in F-G loops.
Torimoto
Nao
N
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8675, Japan.
Ishii
Itsuko
I
Toyama
Ken-Ichi
K
Hata
Masayuki
M
Tanaka
Kanako
K
Shimomura
Hitoshi
H
Nakamura
Hiroyoshi
H
Ariyoshi
Noritaka
N
Ohmori
Shigeru
S
Kitada
Mitsukazu
M
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D019314:Dehydroepiandrosterone Sulfate / Q000378:metabolism; D006900:Hydroxylation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity
D050506:Mutant Chimeric Proteins; D003687:Dehydroepiandrosterone; D019314:Dehydroepiandrosterone Sulfate; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human