Helices F–G Are Important for the Substrate Specificities of CYP3A7

Abstract

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...

Comparison of the substrate kinetics of pig CYP3A29 with pig liver microsomes and human CYP3A4

“…When compared, the amino acid sequences of the two enzymes with respect to substrate recognition region, the most distinct differences observed were located in the helices F-G region (see Supplementary Figure S3 available at http://www.bioscirep.org/bsr/031/bsr0310211add.htm). Helices F-G were considered as an important region for determining the substrate specificities of CYP3A enzymes [43] due to their function in initial substrate recognition [41,44]. This region contains a number of residues that have been shown by site-directed mutagenesis to have a direct or indirect role in CYP3A4 function.…”

Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus

“…[ 3 H]16␣-OH DHEAS was generated by incubating [1,2,6,7-3 H(N)]-dehydroepiandrosterone sulfate, sodium salt ([ 3 H]D-HEAS, 79.5 Ci/mmol; PerkinElmer) with CYP3A7 supersomes supplemented with P450 reductase and cytochrome b5 (BD Gentest) in nicotinamide adenine dinucleotide phosphate regeneration system solution (1.3 mM oxidized nicotinamide adenine dinucleotide phosphate, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl 2 , and 0.4 U/mL glucose-6-phosphate dehydrogenase in 50 M sodium citrate buffer) (BD Gentest) at 37°C for 2 hours, as described previously (18). Thereactionwasterminatedwithmethanol,followed by centrifugation at 14 000 ϫ g. The supernatant was filtered through a 0.45-m syringe filter (Millex-LH; Millipore).…”

CYP3A4 Catalytic Activity Is Induced in Confluent Huh7 Hepatoma Cells

“…However, it is usually expressed at much lower levels in the liver and is only detectable in 20 to 30% of the human population (Guengerich, 1999;Eichelbaum and Burk, 2001). CYP3A7 is 90% homologous with CYP3A4 (Torimoto et al, 2007) and accounts for at least 50% of the total P450 in human fetal liver but is seldom expressed in the adult liver (Eichelbaum and Burk, 2001). CYP3A43 is the most recently discovered isoform (Domanski et al, 2001) and is expressed at approximately 0.1% of CYP3A4 expression levels in the liver, sharing approximately 90% homology (Westlind et al, 2001).…”