Tailoring toluene para-monooxygenase of ralstonia pickettii PKO1 for regiospecific oxidation of aromatics using active site engineering

A. Fishman, Y. Tao, W. E. Bentley, T. K. Wood

Research output: Contribution to conferencePaperpeer-review


Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, formerly known as toluene 3-monooxygenase, has been shown recently by our group to perform primarily para hydroxylation of monosubstituted benzenes (rather than meta) producing 90% p-cresol and 10% m-cresol from toluene oxidation and successively transforming them into 4-methylcatechol (J. Bacterial. 186:3117, 2004). Here, using protein engineering at the α- subunit of the hydroxylase, TbuA1, a double mutant I100S/G103S was constructed, capable of producing 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene, thus exhibiting for the first time true meta-hydroxylation capabilities for a toluene monooxygenase. One ortho TbuA1 variant was created, A107G, which oxidized toluene to mainly o-cresol (80%), methoxybenzene to o-methoxyphenol (88%), and naphthalene to 97% 1-naphthol, all of which are comparable regiospecificities with toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4. A para TbuA1 variant, A107T, produced only p-cresol (>98%), p-methoxyphenol (>99%), and p-nitrophenol (>99%) from toluene, methoxybenzene, and nitrobenzene respectively, exceeding T4MO of P. mendocina KR1 in its complete regiospecificity. Thus, using saturation and site-specific mutagenesis at positions I100, G103, and A107 we have constructed variants of TpMO with all possible regiospecificities for ring-hydroxylation of toluene (ortho, meta, and para positions) and naphthalene; this is the first report of the transformation of a single enzyme into all possible regiospecific variants. Furthermore, we have found that these positions influence regiospecific changes in hydroxylation of substituted phenols. Five previously-uncharacterized wild-type TpMO substrates o-cresol, m-cresol, p-cresol, guaiacol, and m-nitrophenol were identified, and TpMO TbuA1 variants I100S, G103S, I100S/G103S, A107G, and A107T were identified which produce four novel, industrially-significant products (methylhydroquinone, methoxyhydroquinone, 4-methylcatechol, and 4-nitrocatechol).

Original languageEnglish (US)
Number of pages1
StatePublished - 2004
Event2004 AIChE Annual Meeting - Austin, TX, United States
Duration: Nov 7 2004Nov 12 2004


Other2004 AIChE Annual Meeting
Country/TerritoryUnited States
CityAustin, TX

All Science Journal Classification (ASJC) codes

  • General Engineering


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