Engineering cytochrome P-450s: chimeric enzymes.
Cytochrome P-450 isozymes represent a critical component of nature's spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5alpha was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.
Sukumaran
Suja
S
National Environmental Engineering Research Institute, Nehru Marg, Nagpur, India.
Atkins
William Mark
WM
Shanker
Rishi
R
D000595:Amino Acid Sequence; D019475:Camphor 5-Monooxygenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods
D011993:Recombinant Fusion Proteins; D019392:Cytochrome P-450 CYP2E1; D019475:Camphor 5-Monooxygenase