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J. Biol. Chem., Vol. 280, Issue 42, 35382-35390, October 21, 2005

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Molecular Characterization of the Gallate Dioxygenase from Pseudomonas putida KT2440

THE PROTOTYPE OF A NEW SUBGROUP OF EXTRADIOL DIOXYGENASES^*

Juan Nogales, Ángeles Canales, Jesús Jiménez-Barbero, José Luis García, and Eduardo Díaz1

From the Departamento de Microbiología Molecular and Estructura de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain

In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe²⁺ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the K_m and V_max values for gallate were 144 µM and 53.2 µmol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1–281) and C-terminal domain (residues 294–420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.

Received for publication, March 8, 2005 , and in revised form, June 21, 2005.

^* This work was supported by European Union Contract QLRT-2001-02884 and Grants GEN2001-4698-C05-02 and BIO2003-05309-C04-02 from the Comisión Interministerial de Ciencia y Tecnología. 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.

¹ To whom correspondence should be addressed: Dept. de Microbiología Molecular, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. Tel.: 34-918373112; Fax: 34-915360432; E-mail: ediaz{at}cib.csic.es.

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