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J. Biol. Chem., Vol. 279, Issue 47, 48630-48639, November 19, 2004

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The Role of the Conserved Residues His-246, His-199, and Tyr-255 in the Catalysis of Catechol 2,3-Dioxygenase from Pseudomonas stutzeri OX1^*

Ambra Viggiani, Loredana Siani, Eugenio Notomista, Leila Birolo¶, Piero Pucci¶, and Alberto Di Donato||

From the Dipartimento di Chimica Biologica, Università di Napoli Federico II, Via Mezzocannone 16, 80134 Napoli, Italy and the ^¶Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Via Cinthia, 80126 Napoli, Italy

Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.

Received for publication, June 4, 2004 , and in revised form, August 31, 2004.

^* 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 supplemental material (including Table S1) that elaborates on the reaction scheme in Fig. 3.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ496739.

These authors contributed equally to this work.

^|| To whom correspondence should be addressed. Tel.: 39-081-674426; Fax: 39-081-674414; E-mail: didonato{at}unina.it.

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