DOI: 10.1046/j.1432-1327.2001.02212.x. Protein engineering of <i>Bacillus megaterium</i> CYP102

Protein engineering of Bacillus megaterium CYP102

10.1046/j.1432-1327.2001.02212.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants. Wild‐type CYP102 (P450BM‐3) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40‐fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min−1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min−1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity. The PAHs were oxidized to a mixture of phenols and quinones. Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10‐phenanthrenequinone, the K‐region oxidation product from phenanthrene, was formed. The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.

Bibliography

Carmichael, A. B., & Wong, L. (2001). Protein engineering of Bacillus megaterium CYP102. European Journal of Biochemistry, 268(10), 3117â3125. Portico.

Authors 2

Angus B. Carmichael (first)
Luet‐Lok Wong (additional)

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Dates

Type	When
Created	22 years, 5 months ago (March 11, 2003, 1:09 p.m.)
Deposited	1 year, 10 months ago (Oct. 31, 2023, 12:32 a.m.)
Indexed	1 month, 3 weeks ago (July 14, 2025, 11:48 p.m.)
Issued	24 years, 3 months ago (May 15, 2001)
Published	24 years, 3 months ago (May 15, 2001)
Published Online	23 years, 8 months ago (Dec. 20, 2001)
Published Print	24 years, 3 months ago (May 15, 2001)

Funders 0

None

BibTeX

@article{Carmichael_2001, title={Protein engineering of Bacillus megaterium CYP102: The oxidation of polycyclic aromatic hydrocarbons}, volume={268}, ISSN={1432-1033}, url={http://dx.doi.org/10.1046/j.1432-1327.2001.02212.x}, DOI={10.1046/j.1432-1327.2001.02212.x}, number={10}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={Carmichael, Angus B. and Wong, Luet‐Lok}, year={2001}, month=may, pages={3117–3125} }

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