title: Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression.
abstract: Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located.
authors.affiliation: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of 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, Republic of Korea.
authors.forename: Sukmo
authors.initials: S
authors.lastname: Kang
authors.affiliation: School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea.
authors.forename: Mi Sook
authors.initials: MS
authors.lastname: Dong
authors.affiliation: College of Medicine, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Jung-Duck
authors.initials: JD
authors.lastname: Park
authors.affiliation: College of Agriculture of Life Science, Seoul National University, Seoul, Republic of Korea.
authors.forename: Jinseo
authors.initials: J
authors.lastname: Park
authors.affiliation: College of Agriculture of Life Science, Seoul National University, Seoul, Republic of Korea.
authors.forename: Sangkee
authors.initials: S
authors.lastname: Rhee
authors.affiliation: College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea. Electronic address: dyryu@snu.ac.kr.
authors.forename: Doug-Young
authors.initials: DY
authors.lastname: Ryu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D016466:CHO Cells; D003001:Cloning, Molecular; D003412:Cricetulus; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006801:Humans; D006900:Hydroxylation; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010078:Oxazines / Q000378:metabolism; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D010078:Oxazines; D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D006418:Heme; C007649:ethoxyresorufin; D019363:Cytochrome P-450 CYP1A1
keywords: CYP1A1; CYP1A1*12; Enzyme activity; Heme; Protein structure; α-helix