Biochemical analysis of recombinant CYP4A11 allelic variant enzymes: W126R, K276T and S353G

Drug Metab Pharmacokinet. 2016 Dec;31(6):445-450. doi: 10.1016/j.dmpk.2016.09.003. Epub 2016 Sep 19.

Abstract

Human CYP4A11 is the major ω-hydroxylase of fatty acids in the liver and kidneys. It produces 20-hydroxyeicosatetraenoic acid as well as hydroxylates fatty acids. In this study, we investigated the biochemical properties of three alleles of CYP4A11: W126R, K276T, and S353G. Site-directed mutagenesis of the wild type CYP4A11 was performed, to construct the W126R, K276T, and S353G variant clones. The CYP4A11 wild type and variant constructs were heterologously expressed in Escherichia coli. CO-binding spectra showed the expression of the wild type, K276T and S353G variants, indicating the functional P450 holoenzyme. The W126R variant was not expressed in E. coli. Binding affinities of lauric acid in K276T and S353G variants were stronger than that of wild type. Steady-state kinetics in the hydroxylation reaction of fatty acids were studied. The catalytic efficiencies (kcat/Km) of K276T and S353G variants in the reactions without cytochrome b5 were approximately 2- and 4-fold higher, respectively, than that of wild type, and in the reactions with cytochrome b5 they were approximately 2- and 3-fold higher, respectively. These results suggest that individuals carrying the alleles, K276T and S353G, might exhibit higher catalysis of CYP4A11, which may affect the endogenous metabolic products associated with regulation of blood pressure.

Keywords: Allele; CYP4A11; Fatty acid hydroxylase; P450; Polymorphism.

MeSH terms

  • Alleles*
  • Biocatalysis
  • Cytochrome P-450 CYP4A / chemistry
  • Cytochrome P-450 CYP4A / genetics*
  • Cytochrome P-450 CYP4A / isolation & purification
  • Cytochrome P-450 CYP4A / metabolism*
  • Fatty Acids / metabolism
  • Humans
  • Hydroxylation
  • Polymorphism, Single Nucleotide
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Fatty Acids
  • Recombinant Proteins
  • CYP4A11 protein, human
  • Cytochrome P-450 CYP4A