title: Characterization of the Gly45Asp variant of human cytochrome P450 1A1 using recombinant expression.
abstract: Cytochrome P450 1A1 (CYP1A1) is a heme-containing enzyme involved in metabolism of xenobiotics. CYP1A1 containing a Gly45Asp substitution has not yet been characterized. Escherichia coli expressing the Gly45Asp variant, as well as the purified variant protein, had lower CYP (i.e., holoenzyme) contents than their wild-type (WT) equivalents. The purified variant protein had reduced heme contents compared with their WT equivalents. Enhanced supplementation of a heme precursor during culture did not increase CYP content in E. coli expressing the variant, but did for the WT. Substitution of Gly45 with other residues, especially those having large side chains, decreased CYP contents of E. coli expressing the variants to a considerable extent. A 3D structure of CYP1A1 indicates that Gly45, along with other residues of the PR region, interacts with Arg77 of β- strand 1-1, which indirectly interacts with heme. Substitution analyses suggest the importance of residues of the PR region and Arg77 in holoenzyme expression. E. coli membrane and mammalian microsomes expressing the Gly45Asp variant, as well as the purified variant protein, had reduced ethoxyresorufin O-dealkylation activities, compared with the WT equivalents. These findings suggest the Gly45Asp substitution results in a structural disturbance of CYP1A1, reducing its holoenzyme formation and catalytic activity.
authors.affiliation: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, 151-742, South Korea.
authors.forename: Seung Heon
authors.initials: SH
authors.lastname: Lee
authors.affiliation: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, 151-742, South Korea.
authors.forename: Hee Jeong
authors.initials: HJ
authors.lastname: Yu
authors.affiliation: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, 151-742, South Korea.
authors.forename: Seungwoo
authors.initials: S
authors.lastname: Lee
authors.affiliation: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, 151-742, South Korea. Electronic address: dyryu@snu.ac.kr.
authors.forename: Doug-Young
authors.initials: DY
authors.lastname: Ryu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; C569463:CYP1A1 protein, human; D019363:Cytochrome P-450 CYP1A1
keywords: CYP1A1; CYP1A1*13; Gly45Asp; Heme; Holoenzyme