Tailoring Toluene para-Monooxygenase of Ralstonia pickettii PKO1 for Regiospecific Oxidation of Aromatics Using Active Site Engineering

for AICHE 2004 [15C12] Advances in Biocatalysis and Protein Engineering Tailoring Toluene para-Monooxygenase of Ralstonia pickettii PKO1 for Regiospecific Oxidation of Aromatics Using Active Site Engineering A. Fishman, Y. Tao, W. E. Bentley, and T. K. Wood University of Connecticut, Storrs, CT University of Maryland, College Park, MD 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. Bacteriol. 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 ringhydroxylation 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 previouslyuncharacterized 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).