Interaction of gammaxene with site specific mutants of cytochrome P450_cam

438-446Cytochrome P450cam (CYP101) from soil bacteria Pseudomonas putida, is one of the most well studied heme-b monoxygenase. A large number of X-ray crystal structures of this enzyme and its mutants are now available with different types of substrates that can be used to study the topology of the active site of the enzyme. We have a continuing interest in applying the current knowledge of cytochrome P450cam-substrate recognition to rationally design the enzyme for the biotransformation of unnatural substrates, like gammaxene, with the long-term aim of applications in bioremediation of environmental contaminants. Comparison of the structure of target substrate with that of camphor, the natural substrate, led us to engineer the heme active-site and we have found that binding affinities significantly are increased for Y96F, Y96F/L244A and Y96F/T101V mutants. This has shown the way to new functions of the enzymes, which not only has provided a novel approach to the study of the mechanism of this complex super-family of enzymes, but has also led to the discovery of green biocatalysts for environmental applications