Expanding the substrate scope of nitrating cytochrome P450 TxtE by active site engineering of a reductase fusion.

Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of L-tryptophan at room temperature using NO, O 2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the alpha-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significantly alterations to the aromatic and aminoethyl moieties.