2cp4
From Proteopedia
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CRYSTAL STRUCTURE OF THE CYTOCHROME P450-CAM ACTIVE SITE MUTANT THR252ALA
Overview
The crystal structure of a cytochrome P-450CAM site-directed mutant in, which the active site Thr252 has been replaced with an Ala (Thr252Ala) has, been refined to an R factor of 0.18 at 2.2 A. According to sequence, alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among, P-450 enzymes. The crystallographic structure of ferrous camphor- and, carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252, is a key active site residue, forming part of the dioxygen-binding site., Mutation of the active site threonine to alanine produces an enzyme in, which substrate hydroxylation is uncoupled from electron transfer., Specifically, hydrogen peroxide and "excess" water are produced instead of, the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a, local distortion in the distal helix between Gly248 and Thr252 becomes, even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule, not present in the native enzyme is positioned in the dioxygen-binding, region of the mutant enzyme active site. In this location, the solvent, molecule could sterically interfere with and destabilize dioxygen binding., In addition, the active site solvent molecule is connected, via a network, of hydrogen bonds, with an internal solvent channel which links distal, helix residues to a buried Glu side chain. Thus, solvent protons appear to, be much more accessible to dioxygen in the mutant than in the wild-type, enzyme, a factor which may promote hydrogen peroxide and/or water, production instead of substrate hydroxylation. On the basis of, crystallographic and mutagenesis data, a proton delivery pathway involving, residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type, P-450CAM. Coordinates of structures discussed in this paper have been, submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).
About this Structure
2CP4 is a Single protein structure of sequence from Pseudomonas putida with HEM and CAM as ligands. Active as Camphor 5-monooxygenase, with EC number 1.14.15.1 Full crystallographic information is available from OCA.
Reference
Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala., Raag R, Martinis SA, Sligar SG, Poulos TL, Biochemistry. 1991 Dec 3;30(48):11420-9. PMID:1742281
Page seeded by OCA on Wed Nov 21 09:11:58 2007
