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J. Biol. Chem., Vol. 279, Issue 29, 30600-30610, July 16, 2004

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Crystal Structure of the Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas stutzeri OX1

INSIGHT INTO THE SUBSTRATE SPECIFICITY, SUBSTRATE CHANNELING, AND ACTIVE SITE TUNING OF MULTICOMPONENT MONOOXYGENASES^*

Matthew H. Sazinsky**, Joel Bard, Alberto Di Donato¶, and Stephen J. Lippard||

From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, the Division of Structural Biology, Wyeth Pharmaceuticals, Cambridge, Massachusetts 02140, and the ^¶Dipartimento di Chimica Biologica, Universita' di Napoli Federico II, 80134 Napoli, Italy

The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regio- and enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 Å or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the ₂₂₂ tertiary structure are notably different. Of particular interest is a 6–10 Å-wide channel of 35 Å in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.

Received for publication, January 22, 2004 , and in revised form, April 13, 2004.

The atomic coordinates and structure factors (codes 1T0R, 1T0Q, and 1T0S) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This research was supported in part by National Institutes of Health Grant GM32134 (to S. J. L.) and the Italian Ministry of University and Research, PRIN 2000 and PRIN 2002 (to A. D. D.). 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Materials.

^** Trainee under Biotechnology Training Grant GM08334.

^|| To whom correspondence should be addressed. Tel.: 617-253-1892; Fax: 617-258-8150; E-mail: lippard{at}lippard.mit.edu.

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