2h7s

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L244A mutant of Cytochrome P450cam

Overview

We have identified a P450(cam) mutation, L244A, that mitigates the, affinity for imidazole and substituted imidazoles while maintaining a high, affinity for the natural substrate camphor. The P450(cam) L244A crystal, structure solved in the absence of any ligand reveals that the I-helix is, displaced inwards by over 1 A in response to the cavity created by the, change from leucine to alanine. Furthermore, the crystal structures of, imidazole-bound P450(cam) and the 1-methylimidazole-bound P450(cam) L244A, mutant reveal that the ligands have distinct binding modes in the two, proteins. Whereas in wild-type P450(cam) the imidazole coordinates to the, iron in an orientation roughly perpendicular to the plane of the heme, in, the L244A mutant the rearranged I helix, and specifically residue Val247, forces the imidazole into an orientation almost parallel to the heme that, impairs its ability to coordinate to the heme iron. As a result, the, imidazole is much more weakly bound to the mutant than it is to the, wild-type enzyme. Despite the constriction of the active site by the, mutation, previous work with the L244A mutant has shown that it oxidizes, larger substrates than the wild-type enzyme. This paradoxical situation, in which a mutation that nominally increases the active site cavity, appears to decrease it, suggests that the mutation actually increases the, active site maleability, allowing it to better expand to oxidize larger, substrates.

About this Structure

2H7S is a Single protein structure of sequence from Pseudomonas putida with HEM as ligand. Active as Camphor 5-monooxygenase, with EC number 1.14.15.1 Full crystallographic information is available from OCA.

Reference

Cytochrome P450 active site plasticity: attenuation of imidazole binding in cytochrome P450(cam) by an L244A mutation., Verras A, Alian A, de Montellano PR, Protein Eng Des Sel. 2006 Nov;19(11):491-6. Epub 2006 Aug 30. PMID:16943206

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