title: A two-component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate in Pseudomonas putida.
abstract: 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).
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, 24007 León, Spain.
authors.forename: Elsa
authors.initials: E
authors.lastname: Arias-Barrau
authors.forename: Angel
authors.initials: A
authors.lastname: Sandoval
authors.forename: Germán
authors.initials: G
authors.lastname: Naharro
authors.forename: Elías R
authors.initials: ER
authors.lastname: Olivera
authors.forename: José M
authors.initials: JM
authors.lastname: Luengo
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002384:Catalysis; D003470:Culture Media / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006713:Homogentisic Acid / Q000737:chemistry / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism / Q000502:physiology*; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010648:Phenylacetates / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D011551:Pseudomonas fluorescens / Q000378:metabolism; D016958:Pseudomonas putida / Q000378:metabolism*; D013997:Time Factors
chemical_list: C026245:3-hydroxyphenylacetate; D003470:Culture Media; D010648:Phenylacetates; D006899:Mixed Function Oxygenases; D006713:Homogentisic Acid