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  • Title: The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2.
    Author: Coleman T, Chao RR, De Voss JJ, Bell SG.
    Journal: Biochim Biophys Acta; 2016 Jun; 1864(6):667-675. PubMed ID: 26969786.
    Abstract:
    BACKGROUND: The 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. METHODS: In vitro substrate binding and kinetic turnover assays coupled with HPLC and GC-MS analysis and whole-cell oxidation turnovers. RESULTS: Modification 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. CONCLUSION: Substrate 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 SIGNIFICANCE: An 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.
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