ABSTRACT: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 16␣-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 16␣-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.Cytochromes P450s (P450s) comprise a superfamily of monooxygenases that metabolize a wide variety of exogenous chemicals as well as endogenous substrates such as steroids, fatty acids, and prostaglandins. Multiple forms of P450 have been shown to be present in the liver (Nelson et al., 1996). Among the forms of P450s, CYP3A is the most abundant form in human livers (Kitada et al., 1985a;Shimada et al., 1994), and the human CYP3A subfamily consists of four isoforms: CYP3A4 (Beaune et al., 1986), CYP3A5 (Aoyama et al., 1989), CYP3A7 (Komori et al., 1989), and CYP3A43 (Domanski et al., 2000.CYP3A7 accounts for 30 to 50% of total P450s in the fetal liver (Kitada et al., 1985b;Shimada et al., 1996). CYP3A4 is the major form of P450 expressed in the adult liver, and the expression of CYP3A4 mRNA was not seen in human fetal livers and began to increase after birth (Lacroix et al., 1997). It has been reported that a 5Ј-flanking region from 1 kilobase to the transcriptional start site of the CYP3A7 gene was 91% identical to that of the CYP3A4 gene (Itoh et al., 1992;Hashimoto et al., 1993). Recently, an experiment using HepG2 cells showed that Sp1 and Sp3 bound to the nuclear factor-B-like element of the CYP3A7, but not the CYP3A4 gene, and that the expression of the CYP3A7 gene was cooperatively regulated by Sp1, Sp3, hepatocyte nuclear factor 3␤, and upstream stimulatory factor 1 (Saito et al., 2001).The nucleotide and amino acid sequences of CYP3A7 (Komori et al., 1989) share 94 and 88% identities, r...