title: Isotopic labeling of the heme cofactor in cytochrome P450 and other heme proteins.
abstract: A recombinant bacterial expression system that generates (13)C-labeled heme or (15)N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which the HemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with (13)C or (15)N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, along with one peroxidase. Confirmed both (13)C- and (15)N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.
authors.affiliation: The Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, 416 Sullivan Science Building, Greensboro, NC 27402, USA.
authors.forename: David
authors.initials: D
authors.lastname: Bryson
authors.forename: Pek-Lang
authors.initials: PL
authors.lastname: Lim
authors.forename: Alin
authors.initials: A
authors.lastname: Lawson
authors.forename: Sandhya
authors.initials: S
authors.lastname: Manjunath
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Raner
mesh_terms: D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D006418:Heme; D000622:Aminolevulinic Acid; D003577:Cytochrome P-450 Enzyme System