title: Genotyping and site-directed mutagenesis of a cytochrome P450 meander Pro-X-Arg motif critical to CYP4B1 catalysis.
abstract: CYP4B1 isoforms from rodents and other common laboratory animals are involved in the bioactivation of a range of protoxins, including 2-aminofluorene, 4-ipomeanol, and valproic acid. However, an earlier study provided evidence for a human allele encoding a nonfunctional CYP4B1 enzyme due to a Pro427Ser transversion in the meander region of the protein. In the present study, the CYP4B1 gene from several racial groups, Caucasians, African-Americans, and Hispanics, and from six nonhuman primate species was genotyped using a PCR-Hinf1 restriction enzyme fragment length polymorphism assay or by direct sequencing. All human populations examined were found to possess only the Ser allele at codon 427 ((1279)TCT) and all of the nonhuman primate species possessed only the Pro (CCT) allele. Therefore, an inactivating (1279)C-->T mutation in the human CYP4B1 gene likely arose following divergence of the Homo and Pan clades. Amino acid sequence alignments revealed further that this key Pro residue is located two amino acid residues N-terminal to the distal Arg of a Glu-Arg-Arg triad thought to participate in heme binding and/or redox partner interactions. Mutation of the corresponding Arg424 residue in rabbit CYP4B1 to Leu, but not His, resulted in a loss of lauric acid hydroxylase activity and ability to generate a reduced-CO binding spectrum. These data provide additional evidence for the importance of this meander region Pro-X-Arg motif in CYP4B1 heme binding and catalytic function.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: Zheng
authors.forename: Kirk R
authors.initials: KR
authors.lastname: Henne
authors.forename: Patrick
authors.initials: P
authors.lastname: Charmley
authors.forename: Richard B
authors.initials: RB
authors.lastname: Kim
authors.forename: D Gail
authors.initials: DG
authors.lastname: McCarver
authors.forename: Erwin T
authors.initials: ET
authors.lastname: Cabacungan
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Hines
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D002384:Catalysis; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005838:Genotype; D006801:Humans; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011323:Primates; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1; D042926:Cytochrome P-450 CYP4A