The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

• CYP199A4 has high selectivity for the carboxylate group of 4-methoxybenzoic acid.
• This selectivity arises from interactions with arginine and serine residues.
• Alternative functional groups can bind to CYP199A4 but with low affinity.
• The activities of the in vitro enzyme assays can be extrapolated to whole-cell oxidation systems.
• The high regioselectivity for oxidation at the para-substituent is maintained.

BackgroundThe cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic acid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This occurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids.MethodsIn vitro substrate binding and kinetic turnover assays coupled with HPLC and GC–MS analysis and whole-cell oxidation turnovers.ResultsModification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid.ConclusionSubstrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4.General significanceAn understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates.