Rational approach to improving reductive catalysis by cytochrome P450cam.
Although halogenated hydrocarbons are noted for low chemical reactivity, small amounts are toxic to humans. Cytochromes P450 have been implicated in transforming these compounds to more reactive species, under anaerobic conditions, through reduction at the heme. A significant amount of effort has been directed toward turning this catalytic ability to our advantage by engineering P450 variants than can efficiently remediate these compounds in situ, before they come in contact with the human population. We have taken a 'rational' approach to this problem, in which a combination of theory and molecular modeling is applied to identify which properties of the enzyme have the greatest influence over reductive dehalogenation. Recent progress in this area is briefly reviewed. Two novel mutants, incorporating tryptophan (positions 87 and 396) and histidine (position 96, neutral and protonated) amino acid substitutions in the active site, are proposed and evaluated using molecular dynamics simulations. The upper bound on rate enhancement relative to wild-type is estimated in each mutant using electron transfer theory. The most significant rate enhancement is predicted for the His 96 mutant in the protonated state; while some His residues of certain proteins exhibit a pKa high enough to support a large protonated population, such information is not presently available for this proposed mutant.
Manchester
J I
JI
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Ornstein
R L
RL
D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003198:Computer Simulation; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D010084:Oxidation-Reduction
D019475:Camphor 5-Monooxygenase