DESIGN OF A NOVEL P450 - A FUNCTIONAL BACTERIAL-HUMAN CYTOCHROME-P450CHIMERA

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membra ne-bound) cytochrome P450 2C9. The chimeric protein shows a reduced GO -difference spectrum absorption at 446 nm, and circular dichroism spec tra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin re ductase. This chimera provides a novel method for addressing structure -function issues and may prove useful in the design of oxidants for be nign and stereospecific synthesis, as well as catalysts for bioremedia tion of polluted areas. Furthermore, these results provide the first e vidence that bacterial P450 enzymes and mammalian P450 enzymes are lik ely to share a common tertiary structure.