Protein engineering of toluene 4-monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4-nitrocatechol from nitrobenzene

Biotechnology and Bioengineering
Ayelet FishmanThomas K Wood

Abstract

After discovering that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4-nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis. Screening 550 colonies from a random mutagenesis library generated by error-prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol-producing mutants. One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, I84Y, S95T, I100S, S400C; TmoB D79N). It was believed that position I100 of the alpha subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho-monooxygenase of Burkholderia cepacia G4. Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4-nitrocatechol formation by I100A was more than 16 times higher than that of wild-type T4MO at 200 microM nitrobenzene (0.13 +/- 0.01 vs. 0.008 +/- 0.001 nmol/min.mg protein). HPLC and mass spectrometry analysis revealed that variants NB1, I100A, and I100S produce 4-nitrocatechol via m-...Continue Reading

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Citations

Aug 25, 2009·Nature Chemical Biology·Martina PavlovaJiri Damborsky
Feb 28, 2013·Protein Engineering, Design & Selection : PEDS·Janna ShainskyAyelet Fishman
Jul 8, 2009·Applied and Environmental Microbiology·Viviana Sanchez-TorresThomas K Wood
Aug 17, 2010·Applied and Environmental Microbiology·Moran BroukAyelet Fishman
Jan 15, 2008·Applied and Environmental Microbiology·Roi FeingerschAyelet Fishman
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