title: Site-specific fluorescent labeling and oriented immobilization of a triple mutant of CYP3A4 via C64.
abstract: The generation of site-specific bioconjugates of proteins is highly desired for a number of biophysical and nanotechnological applications. To this end, many strategies have been developed that allow the specific modification of certain canonical amino acids and, more recently, noncanonical functional groups. P450 enzymes are heme-dependent monooxygenases involved in xenobiotic metabolism and in the biosynthesis of a variety of secondary metabolites. We became interested in the site-specific modification of these enzymes, CYP3A4 in particular, through our studies of their in vitro biocatalytic properties and our desire to exploit their remarkable ability to oxidize unactivated C-H bonds in a regio- and stereospecific manner. Obtained via a partial cysteine-depletion approach, a functional triple mutant of CYP3A4 (C98S/C239S/C468G) is reported here which is singly modified at C64 by maleimide-containing groups. While cysteine-labeling of the wild-type enzyme abolished >90% of its enzymatic activity, this mutant retained ≥75% of the activity of the unmodified wild-type enzyme with 9 of the 18 maleimides that were tested. These included both fluorescent and solid-supported maleimides. The loss of activity observed after labeling with some maleimides is attributed to direct enzyme inhibition rather than to steric effects. We also demonstrate the functional immobilization of this mutant on maleimide-functionalized agarose resin and silica microspheres.
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montréal, Québec, Canada, H3A 0B8.
authors.forename: Amélie
authors.initials: A
authors.lastname: Ménard
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.forename: Pierre
authors.initials: P
authors.lastname: Karam
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Cosa
authors.forename: Karine
authors.initials: K
authors.lastname: Auclair
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D008863:Microspheres; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D012822:Silicon Dioxide / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D008301:Maleimides; D050505:Mutant Proteins; C043592:maleimide; D012822:Silicon Dioxide; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003545:Cysteine