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Journal of Bacteriology, May 2004, p. 3117-3123, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.3117-3123.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para-Hydroxylating Enzyme

Ayelet Fishman, Ying Tao, and Thomas K. Wood*

Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3222

Received 12 January 2004/ Accepted 9 February 2004

Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ± 0.33 nmol/min/mg of protein with an apparent Km value of 250 µM and produced 90% p-cresol and 10% m-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p-cresol and 3% m-cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.

* Corresponding author. Mailing address: Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3222. Phone: (860) 486-2483. Fax: (860) 486-2959. E-mail: twood{at}engr.uconn.edu.

Journal of Bacteriology, May 2004, p. 3117-3123, Vol. 186, No. 10
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.10.3117-3123.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Feingersch, R., Shainsky, J., Wood, T. K., Fishman, A. (2008). Protein Engineering of Toluene Monooxygenases for Synthesis of Chiral Sulfoxides. Appl. Environ. Microbiol. 74: 1555-1566 [Abstract] [Full Text]  
  • Vardar, G., Wood, T. K. (2005). Alpha-Subunit Positions Methionine 180 and Glutamate 214 of Pseudomonas stutzeri OX1 Toluene-o-Xylene Monooxygenase Influence Catalysis. J. Bacteriol. 187: 1511-1514 [Abstract] [Full Text]  
  • Fishman, A., Tao, Y., Rui, L., Wood, T. K. (2005). Controlling the Regiospecific Oxidation of Aromatics via Active Site Engineering of Toluene para-Monooxygenase of Ralstonia pickettii PKO1. J. Biol. Chem. 280: 506-514 [Abstract] [Full Text]  
  • Tao, Y., Fishman, A., Bentley, W. E., Wood, T. K. (2004). Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone. J. Bacteriol. 186: 4705-4713 [Abstract] [Full Text]  


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