Cytochrome directed evolution

Gonzalez FJ, Gelboin HV. Human cytochromes P450 evolution and cDNA-directed expression. Environ Health Perspect 1992 98 81-85. 

Iffland A, Gendreizig S, Tafemeyer P, Johnsson K (2001) Changing the substrate specificity of cytochrome c peroxidase using directed evolution. Biochem Biophys Res Commun 286 126-132... 

An example of the use of isolated enzymes is the work on directed evolution by random mutagenesis of the fatty acid hydroxylase cytochrome P450 BM-3 by the group of Arnold [254] (Table 1.10). The target reaction was the enantioselective... 

Metal mediated epoxidahon is remarkably diverse, with many types of ligand systems being represented. For example, a cytochrome P450 BM-3 mutant (139-3) has been developed using directed evolution, which exhibits high activity towards epoxidation of several non-natural substrates. Thus, exposure of styrene 4 to BM-3 variant 139-3 in phosphate buffer containing methanol and NADPH resulted in the quantitative conversion to styrene oxide 5. For terminal aliphatic alkenes, however, allyhc hydroxylation is the predominant process <04T525>. 

Yasutake Y, Fujii Y, Nishioka T, Cheon W-K, Arisawa A, Tamura T (2010) Structural evidence for enhancement of sequential vitamin D-3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D-3 hydroxylase. J Biol Chem 285 31193-31201... 

Kumar S, Halpert JR (2005) Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts. Biochem Biophys Res Commun 338 456 64... 

Lentz, O., Li, Q.S., Schwaneberg, U., Lutz-Wahl, S., Fischer, P., and Schmid, R.D. (2001) Modification of the fatty acid specificity of cytochrome P450 BM-3 from BaciUus megaterium by directed evolution a validated assay. J. Md. Catal B Enzym., 15, 123 133. 

Kumar S, Chen CS, Waxman DJ, Halpert JR (2005) Directed evolution of manunalian cytochrome P450 2B1, mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide. J Biol Chem 280 19569-19575... 

Axarli I, Prigipaki A, Labrou NE (2005) Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution production of an efficient enzyme for bioconversion of fine chemicals. Biomol Eng 22 81-88... 

E. T. Farinas, U. Schwaneberg, A. Glieder, F.H. Arnold, Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation, Adv. Synth. Catal. 343 (2001) 601-606. 

Iffland A, Tafemeyer P, Saudan C, Johnsson K (2000) Directed molecular evolution of cytochrome c peroxidase. Biochemistry 39 10790-10798... 

These screens have been used to direct the evolution of cytochrome P450 BM-3, a soluble enzyme from Bacillus megaterium that contains its reductase and hydroxylase domains on a single polypeptide chain. P450 BM-3 primarily catalyzes the hydroxylation of fatty acids ( 12 to 18 carbons long) at the a>-1, a>-2, and m-3 positions, but also... 

The resemblance among cytochrome c molecules extends to the level of amino acid sequence. Because of the molecule s relatively small size and ubiquity, the amino acid sequences of cytochrome c from more than 80 widely ranging eukaryotic species were determined by direct protein sequencing by Emil Smith, Emanuel Margoliash, and others. Comparison of these sequences revealed that 26 of 104 residues have been invariant for more than one and a half billion years of evolution. A phylogenetic tree, constructed from the amino acid sequences of cytochrome c, reveals the evolutionary relationships between many animal species (Figure 18.24). 

The electrons provided in the light reaction, however, may also be directly exported from the cells and used to reduce a variety of extracellular substrates. This electron export is effected by surface enzymes (called transplasmamembrane reductases) spanning the plasmamembrane from the inside surface to the outside. They transfer electrons from an internal electron donor [chiefly NADH and NADPH see Crane et al. (1985)] to an external electron acceptor. Direct reduction of extracellular compounds by transplasmamembrane electron transport proteins is prevalent in all cells thus far examined (Fig. 2.2). Although the function of this redox system is still subject to speculation, in phytoplankton it shows considerable activity, relative to other biochemical processes. A host of membrane-impermeable substrates, including ferricyanide, cytochrome c, and copper complexes, are reduced directly at the cells surface by electrons originating from within the cell. In phytoplankton, where the source of electrons is the light reactions of photosynthesis, the other half-redox reaction is the evolution of ()2 from H20. In heterotrophs, the electrons originate in the respiration of reduced substances. 

A. Iffland, P. Tafelmeyer, C. Saudan, K. Johansson, Directed Molecular Evolution of Cytochrome c Peroxidase. Biochemistry, 39 (2000) 10790-10798. 

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