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

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, performs 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-methylcatecholf. Using protein engineering at the α-subunit of the hydroxylase, TbuA1, a double mutant 1100S/G103S was constructed, capable of producing 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene. One ortho TbuA1 variant was created, A107G, which oxidized toluene to mainly o-cresol, methoxybenzene to o-methoxyphenol, and naphthalene to 97% 1-naphthol. A para TbuA1 variant, A107T, produced only p-cresol, p-methoxyphenol, and p-nitrophenol 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 1100, G103, and A107 variants of TpMO with all possible regiospecificities for ring hydroxylation of toluene and naphthalene were constructed. Previously uncharacterized wild-type TpMO substrates o-cresol, m-cresol, p-cresol, guaiacol, and m-nitrophenol were identified, and TpMO TbuA1 variants 1100S, G103S, 1100S/G103S, A107G, and A107T were identified which produce four novel, industrially-significant products (methylhydroquinone, methoxyhydroquinone, 4-methylcatechol, and 4-nitrocatechol). This is an abstract of a paper presented at the AIChE Annual Meeting (Austin, TX 11/7-12/2004).