Protein engineering of the cytochrome P450 monooxygenase from bacillus megaterium

Abstract

The role and importance of cytochrome P450 enzymes (CYP) in drug development, biodegradation processes and biocatalysis has been widely acknowledged. P450 monooxygenases exhibit an extremely wide substrate spectrum which is the basis of their ability to activate or detoxify a large variety of target molecules. P450 monooxygenases have been isolated from bacteria, yeasts, insects, as well as mammalian and plant tissues. Currently, the enzyme family is one of the best known gene subfamilies with over 1000 characterized members (http://drnelson.utmem.edu/CytochromeP450.html). Many studies have been dedicated to structural models of cytochrome P450 in order to improve our understanding of the mechanistic details of the enzymes, their substrate specificity and their pronounced stereo- and regiospecificity. In addition, homology modeling of mammalian P450s and QSAR analyses using chemicals which are metabolized by P450s, have added considerably to our understanding of the metabolic variations and functions of the enzyme. Cytochrome P450 enzymes are of considerable interest to pharmaceutical and chemical industry and have thus become targets for protein engineering approaches. Protein engineering is generally defined as the modification of an enzyme by site-directed or random mutagenesis with the aim of altering its properties. Rational design requires a solid structural basis and profound knowledge of the catalytic mechanism of the enzyme which was provided by determining the structures of CYPs using X-ray crystallography at high-resolution. Ten of the twelve crystallized cytochrome P450s are of prokaryotic origin and water-soluble. From a technical point of view, microbial P450s are easier to handle than P450 enzymes from plants and animals. They are not membrane-associated and exhibit a relatively high stability. Eukaryotic cytochrome P450 enzymes are membrane-associated proteins and are hence more difficult to crystallize. Currently, only the X-ray structures of two membrane-bound mammalian P450s, rabbit CYP2C5 and human CYP2C9 are known. Models of other mammalian P450s were built based on the structure of CYP2C5 and its bacterial analogues. P450cam, the cytochrome P450 monooxygenase from Pseudomonas putida, is the best characterized microbial P450 enzyme. In the last fifteen years, a large number of other soluble prokaryotic P450 enzymes have been identified, isolated, subcloned in Escherichia coli, overexpressed and characterized. Cytochrome P450 BM-3 from Bacillus megaterium is catalytically self-sufficient. It contains a P450-heme domain of 54 kDa and an FAD/FMN-reductase domain of 64kDa on a single polypeptide chain. The enzyme catalyzes the subterminal oxidation of saturated and unsaturated fatty acids with a chain length of 12 to 20 carbons. High-resolution X-ray crystal structures are available for substrate-free, palmitic acid-bound and N-palmitoylglycine-bound wild-type and mutant P450 BM-3 enzymes. The structure resolved by NMR is also available. The well-known structure, the availability of the CYP102A1 gene which encodes the protein, and the possibility of expressing the protein in E. coli have encouraged a number of research groups to undertake site-directed mutagenesis studies in order to identify key amino acids. Insights into the mechanisms of P450 BM-3 have been gained and transferred to eukaryotic P450 enzymes. Since P450 BM-3 is an excellent model for addressing questions on the wide substrate specificity of P450s in general and techniques involving the mutagenesis of P450 BM-3 have led to a variety of biocatalysts with features of industrial interests. This review will summarize the recent research on this particular P450 enzyme.