Expansion of substrate specificity of cytochrome P450 2A6 by random and site-directed mutagenesis

J Biol Chem. 2005 Dec 9;280(49):41090-100. doi: 10.1074/jbc.M508182200. Epub 2005 Oct 7.

Abstract

The natural product indole is a substrate for cytochrome P450 2A6. Mutagenesis of P450 2A6 was done to expand its capability in the oxidization of bulky substituted indole compounds, which are not substrates for the wild-type enzyme or the double mutant L240C/N297Q, as determined in our previous work (Wu, Z.-L., Aryal, P., Lozach, O., Meijer, L., and Guengerich, F. P. (2005) Chem. Biodivers. 2, 51-65). Error-prone PCR and site-directed mutagenesis led to the identification of two critical amino acid residue changes (N297Q and I300V) that achieve the purpose. The new mutant (N297Q/I300V) was able to oxidize both 4- and 5-benzyloxy(OBzl)indoles to form colored products. Both changes were required for oxidation of these bulky substrates. The colored product derived from 5-OBzl-indole was mainly 5,5'-di-OBzl-indirubin, whereas the dominant blue dye isolated upon incubations with 4-OBzl-indole was neither an indigo nor an indirubin. Two-dimensional NMR experiments led to assignment of the structure as 4-OBzl-2-(4'-OBzl-1',7'-dihydro-7'-oxo-6'H-indol-6'-ylidene)indolin-3-one, in which a pyrrole ring and a benzene ring are connected with a double bond instead of the pyrrole-pyrrole connection of other indigoids. Monomeric oxidation products were also isolated and characterized; three phenols (4-OBzl-1H-indol-5-ol, 4-OBzl-1H-indol-6-ol, and 4-OBzl-1H-indol-7-ol) and one quinone (4-OBzl-1H-indole-6,7-dione, the postulated immediate precursor of the final blue dye) were identified. The results are interpreted in the context of a crystal structure of a P450 2A6-coumarin complex. The I300V change opens an additional pocket to accommodate the OBzl bulk. The N2297Q change is postulated to generate a hydrogen bond between Gln and the substrate oxygen. Thus, the substrate specificity of P450 2A6 was expanded, and new products were obtained in this study.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aryl Hydrocarbon Hydroxylases / chemistry
  • Aryl Hydrocarbon Hydroxylases / genetics*
  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Benzyl Compounds
  • Binding Sites
  • Coumarins / metabolism
  • Crystallization
  • Cytochrome P-450 CYP2A6
  • Gene Library
  • Humans
  • Hydrogen Bonding
  • Indoles / analysis
  • Indoles / metabolism
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / metabolism*
  • Models, Molecular
  • Mutagenesis, Site-Directed*
  • Oxidation-Reduction
  • Polymerase Chain Reaction
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Benzyl Compounds
  • Coumarins
  • Indoles
  • Recombinant Proteins
  • 4-chloroindole
  • indole
  • coumarin
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP2A6

Associated data

  • PDB/1PQ2