Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.
The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.
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Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, D-70569, Stuttgart, Germany.
Schwaneberg
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Fischer
M
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Schmitt
J
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Pleiss
J
J
Lutz-Wahl
S
S
Schmid
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RD
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015195:Drug Design; D019143:Evolution, Molecular; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002264:Carboxylic Acids; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases