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J. Biol. Chem., Vol. 280, Issue 1, 506-514, January 7, 2005

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Controlling the Regiospecific Oxidation of Aromatics via Active Site Engineering of Toluene para-Monooxygenase of Ralstonia pickettii PKO1^*

Ayelet Fishman, Ying Tao, Lingyun Rui, and Thomas K. Wood

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

A primary goal of protein engineering is to control catalytic activity. Here we show that through mutagenesis of three active site residues, the catalytic activity of a multicomponent monooxygenase is altered so that it hydroxylates all three positions of toluene as well as both positions of naphthalene. Hence, for the first time, an enzyme has been engineered so that its regiospecific oxidation of a substrate can be controlled. Through the A107G mutation in the -subunit of toluene para-monooxygenase, a variant was formed that hydroxylated toluene primarily at the ortho-position while converting naphthalene to 1-naphthol. Conversely, the A107T variant produced >98% p-cresol and p-nitrophenol from toluene and nitrobenzene, respectively, as well as produced 2-naphthol from naphthalene. The mutation I100S/G103S produced a toluene para-monooxygenase variant that formed 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene; thus, for the first time a true meta-hydroxylating toluene monooxygenase was created.

Received for publication, September 8, 2004 , and in revised form, October 18, 2004.

^* This research was supported by National Science Foundation Grant BES-0124401 and by the United States Environmental Protection Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 860-486-2483; Fax: 860-486-2959; E-mail: twood{at}engr.uconn.edu.

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