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J. Biol. Chem., Vol. 280, Issue 28, 26435-26447, July 15, 2005

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A Two-component Hydroxylase Involved in the Assimilation of 3-Hydroxyphenyl Acetate in Pseudomonas putida^*

Elsa Arias-Barrau, Ángel Sandoval¶, Germán Naharro||, Elías R. Olivera, and José M. Luengo**

From the Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, 24007 León and the ^||Departamento de Patología Animal (Sanidad Animal), Facultad de Veterinaria, Universidad de León, 24007 León, Spain

The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, L-Phe, and L-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).

Received for publication, February 22, 2005 , and in revised form, April 21, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY929299, AY929300, and AY937229.

^* This work was supported in part by the Comisión Interministerial de Ciencia y Tecnología, Madrid, Spain, Grant BIO2003-05309-C04-01, and by a Grant of Excma Diputación de León (2005). 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.

Recipient of a fellowship from the Comisión Interministerial de Ciencia y Tecnología.

^¶ Recipient of a fellowship from the University of León.

^** To whom correspondence should be addressed. Tel.: 34-987-291228; Fax: 34-987-291226; E-mail: dbbjlr{at}unileon.es.

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