Comprehensive functional characterization of rare and known CYP2E1 allelic variants identified in a Japanese population.

Cytochrome P450 2E1 (CYP2E1) is a key hepatic enzyme involved in the oxidative metabolism of low-molecular-weight xenobiotics, including drugs, anesthetics, and procarcinogens. Although numerous CYP2E1 genetic variants have been identified, their functional implications remain unclear. In this study, we systematically characterized 25 CYP2E1 variants-22 novel missense variants identified via whole-genome sequencing of 8,380 Japanese individuals, and three PharmVar-defined alleles (*2, *3, and *4). The wild-type and variant enzymes were expressed in 293FT cells, and protein levels were quantified by Western blotting. Enzyme activity was evaluated by measuring chlorzoxazone 6-hydroxylation, and kinetic parameters (Km, Vmax, CLint) were derived from Michaelis-Menten analysis. Five variants exhibited significantly reduced catalytic activity, while two showed increased function. One frameshift variant, Leu447fs, resulted in complete loss of activity. In silico 3D docking simulations using a homology-modeled CYP2E1 structure revealed that several function-altering variants were located near the heme-binding domain or substrate recognition sites. Structural analysis of the Leu133His variant suggested that disruption of hydrogen bonding networks near the heme could underlie its reduced activity. The three known star alleles showed no significant deviation from wild-type activity. These findings provide important mechanistic insights into the functional consequences of rare CYP2E1 variants and underscore the enzyme's structural resilience. Our results offer foundational data for interpreting CYP2E1 genetic variation and support future efforts in personalized medicine through pharmacogenomic profiling.