Download MendeleyInvestigation of the substrate range of CYP199A4: modification of the partition between hydroxylation and desaturation activities by substrate and protein engineering
Bell, S.G., Zhou, R.M., Yang, W., Tan, A.B.H., Gentleman, A.S., Wong, L.-L., Zhou, W.(2012) Chemistry 18: 16677-16688
- PubMed: 23135838
- DOI: https://doi.org/10.1002/chem.201202776
- Primary Citation of Related Structures:
4EGM, 4EGN, 4EGO, 4EGP - PubMed Abstract:
The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4-methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2-naphthoic acid and indole-6-carboxylic acid substrate-bound CYP199A4 complexes reveal the substrate binding modes and the side-chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4-ethylbenzoic acid-bound CYP199A4 revealed that the substrate is held in a similar position to 4-methoxybenzoic acid, and that the C(β) C-H bonds of the ethyl group are closer to the heme iron than those of the C(α) (3.5 vs. 4.8 ?). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4-ethylbenzoic acid desaturase.
Organizational Affiliation:
Department of Chemistry, University of Oxford, UK. stephen.bell@adelaide.edu.au
