The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: structural changes on substrate binding and the identification of an anion binding site

Abstract

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.

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

“…The importance of the individual enzyme-substrate interactions in CYP199A4 can be ascertained from the report that mutation of Ser95 of CYP199A4 to a valine abolished substrate binding while the mutants Arg92Glu and Arg243Thr reduced the binding affinity by at least three orders of magnitude. [22] Therefore the extensive hydrophilic interactions of 4-methoxybenzoic acid with CYP199A4 resulted in the high affinity of the enzyme for this substrate and its selectivity for benzoic acids over even phenylacetic acids. Both the CYP199A2 and A4 enzymes share the structural features which are important for the strong interaction with benzoic acid substrates.…”

“…These enzyme-substrate contacts account for the substrate specificity of the enzyme and mutation of these amino acids dramatically weakened 4-methoxybenzoic acid binding and reduced enzyme activity. [22] These CYP199 enzymes are the only structurally characterised members of this superfamily known to interact strongly with benzoic acid substrates. Other P450 enzymes which are known to interact with this class of substrates include the CYP53 family from fungi which are important drug targets for researchers designing inhibitors as antifungal agents.…”

“…[7,15,22] Ligand binding leads to significant reorientation of the active site and substrate access channel amino acids in order to accommodate the substrate. [22] In the crystal structures of the enzyme bound with benzoic acid substrates (PDB ID: 4DO1, 4EGM and 4EGN), the para-substituent is oriented towards the heme iron while those at the meta-position point away from the heme towards active site amino acids. This is consistent with exclusive attack at the group para to the carboxylate.…”

“…1). [22] In addition, a chloride anion is co-opted by the enzyme. This anion closes off the access channel and also allows the close approach of the two arginine residues, enabling them to interact with the carboxylate group (Fig.…”

“…[9,15,22] with a set of spectra generated from the sum of the appropriate percentages of the spectra of the substrate-free (>95% low-spin, Soret maximum at 418 nm) and camphor-bound (>95% high-spin, Soret maximum at 392 nm) forms of WT CYP101A1.…”