Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation.
Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 +/- 0.3 versus 1.39 +/- 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 +/- 0.03 versus 0.029 +/- 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 +/- 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the alpha-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.
Canada
Keith A
KA
Department of Chemical Engineering, University of Connecticut, Storrs, Connecticut 06269-3222, USA.
Iwashita
Sachiyo
S
Shim
Hojae
H
Wood
Thomas K
TK
D016956:Burkholderia cepacia / Q000201:enzymology / Q000235:genetics; D004000:Dichloroethylenes / Q000378:metabolism*; D019020:Directed Molecular Evolution; D015723:Gene Library; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009284:Naphthols / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism; D014241:Trichloroethylene / Q000378:metabolism*
D004000:Dichloroethylenes; D009284:Naphthols; C029297:vinylidene chloride; D014241:Trichloroethylene; C029350:1-naphthol; D014050:Toluene; D006899:Mixed Function Oxygenases; C093480:toluene ortho-monooxygenase