Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1

FEBS Lett. 2017 Jan;591(1):39-46. doi: 10.1002/1873-3468.12519. Epub 2016 Dec 24.

Abstract

Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP260B1 by site-directed mutagenesis to disrupt the substrate positioning and proton delivery, respectively. Furthermore, we used the kinetic solvent isotope effect on the steady-state turnover of the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.

Keywords: CYP260B1; Sorangium cellulosum So ce56; aldehyde oxidation; kinetic solvent isotope effect.

Publication types

  • Letter

MeSH terms

  • Acrolein / analogs & derivatives*
  • Acrolein / chemistry
  • Acrolein / metabolism
  • Biocatalysis*
  • Cinnamates / metabolism
  • Crystallography, X-Ray
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Deuterium / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Myxococcales / enzymology*
  • Oxidation-Reduction
  • Progesterone / metabolism
  • Protons
  • Solvents
  • Spectrophotometry, Ultraviolet
  • Structure-Activity Relationship
  • Substrate Specificity
  • Threonine / metabolism*

Substances

  • Cinnamates
  • Mutant Proteins
  • Protons
  • Solvents
  • Threonine
  • Progesterone
  • Acrolein
  • Cytochrome P-450 Enzyme System
  • Deuterium
  • cinnamic aldehyde
  • cinnamic acid