Crystal structure of P450cam
(from SWISS-PROT P00183, Camphor 5-monooxygenase), EC 1.14.15.1)
The sequences of hundreds of cytochrome P450 enzymes are now known. The mammalian P450 enzymes are all membrane bound and are involved in a diversity of important physiological processes, ranging from the biosynthesis of sterol hormones to the metabolism of xenobiotics and the unintended activation of many carcinogenic substances. We have been particularly interested in recent years in the CYP4 class of P450 enzymes that catalyze the terminal hydroxylation of fatty acids. In the case of arachidonic acid, the terminally hydroxylated product (20-HETE) appears to play an important role in vasoconstriction.
We have determined that the prosthetic heme group in the CYP4A and CYP4F proteins is covalently bound to the protein via an ester linkage between a glutamic acid residue and a hydroxyl group on one of the methyls of the heme. This completely unexpected finding is unique for the CYP4 class of P450 enzymes and is not found in any other class of P450 enzymes so far examined. Most recently, we have demonstrated by site specific mutagenesis that the covalent link between the protein and the heme is formed through an autocatalytic process (Figure 1) akin to that we have postulated for covalent linking of the heme to the protein in lactoperoxidase (see peroxidase section). Thus, covalent binding occurs when the non-covalently bound heme-protein complex is incubated with NADPH-cytochrome P450 reductase, the normal electron donor partner for the turnover of these enzymes. The details of the covalent binding mechanism, and its significance in terms of enzyme function, are under continuing investigation, as is the development of isoform specific inhibitors of these enzymes.
Figure 1. Proposed partial mechanism for autocatalytic covalent binding of the heme group to the protein in the CYP4 class of P450 enzymes.
In a separate line of investigation, we have cloned, expressed, and characterized the first thermophilic cytochrome P450 enzyme. CYP119 from Sulfolobus sulfotaricus is stable at high temperatures and has been shown to oxidize fatty acids, although the natural substrates for the enzyme are not known. The crystal structure of the enzyme was determined in collaboration with the group of Thomas Poulos at the University of California, Irvine. The structure studies indicate that there is a large rearrangement of the F-G loop when a small ligand (imidazole) is replaced by a larger ligand (phenylimidazole). Protein engineering efforts are underway to define the features that contribute to the thermal stability of CYP119, and to exploit its stability in the design of tailor made catalysts. Efforts to develop a library of tailor made catalysts include work with a variety of other mammalian and bacterial P450 enzymes.
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