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-OHPhAc as the sole carbon source (mhaAB and hmgABC).

Energetics and Surface Properties of Pseudomonas putida DOT-T1E in a Two-Phase Fermentation System with 1-Decanol as Second Phase

“…The major reason for this change in surface properties seems to be an increased adhesion to the surfaces of very poorly watersoluble compounds that leads to an increase in the bioavailability of the compounds (49,50). As in adaptation to poorly water-soluble substrates, uptake systems seem to also be involved, because a hydrophobic surface also works as a kind of source for the compounds that accumulates them at the cell surface and allows better uptake (2). Thus, a more hydrophobic surface can work as a kind of sink for toxic concentrations of solvents that are excluded by efflux pumps but also as a kind of source for less bioavailable substrates that are transported into the cells by several uptake systems.…”

Crystal Structure of the Oxygenase Component (HpaB) of the 4-Hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8

“…This enzyme has been isolated from Escherichia coli (1)(2)(3)(4), Pseudomonas putida (5,6), Klebsiella pneumoniae (7), and Acinetobacter baumannii (8,9). The small component of these enzymes is the flavin reductase, which generates reduced flavin for the oxygenase component to oxygenate substrates.…”

Plasmids containing the same origin of replication are useful tools to perform biotechnological studies in Pseudomonas putida U and in E. coli DH10B

“…In this manuscript, we have analyzed the compatibility/incompatibility of plasmids containing the same origin of replication in the bacteria Pseudomonas putida, and more specifically in P. putida U, a strain with a huge catabolic potential [15][16][17][18][19] and with a remarkable biotechnological interest [20][21][22][23][24].…”