Expanding the Substrate Spectrum of Cytochrome P450 2A6 towards Bulky Substrates*

Abstract

Molecular breeding and biotransformation of cytochrome P450s (P450) are considered two of the emerging areas today. To further expand the substrate spectrum of P450 2A6, N-terminal modification was done to improve its functional expression and consequently, the sensitivity of mutant library screening. Two random libraries screened yielded no gain-of- function mutant, while site-directed mutagenesis generated a new mutant P450 2A6 (N297Q/I300A) with expanded substrate specificity compared with the wild type enzyme and previously reported mutants. The new mutant was able to catalyze the oxidation of 6-benzyloxyindole (6-OBzl-indole) to form colored indigoid dimers as analyzed with mass spectrometry. Both amino acid residue changes were required for the reaction. The results are interpreted in the context of the recently resolved crystal structures of P450 2A6 mutants. The I300A change expands the active-site cavity in both vertical and horizontal directions of the heme plane to be able to fit in substrates of similar nature. The Asn297 is a functionally conserved residue and the N297Q change may be related to hydrogen bond formation.