Isotopic labeling of the heme cofactor in cytochrome P450 and other heme proteins

Biotechnol Lett. 2011 Oct;33(10):2019-26. doi: 10.1007/s10529-011-0661-1. Epub 2011 Jun 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aminolevulinic Acid / metabolism
  • Animals
  • Carbon Isotopes / chemistry
  • Carbon Isotopes / metabolism
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Escherichia coli / metabolism
  • Heme / chemistry*
  • Heme / metabolism
  • Isotope Labeling / methods*
  • Nitrogen Isotopes / chemistry
  • Nitrogen Isotopes / metabolism
  • Protein Isoforms
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Carbon Isotopes
  • Nitrogen Isotopes
  • Protein Isoforms
  • Recombinant Proteins
  • Heme
  • Aminolevulinic Acid
  • Cytochrome P-450 Enzyme System