Purification and characterisation of the NADH: acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWWO of Pseudomonas putida mt‐2

Abstract

The xylene monooxygenase system encoded by the TOL plasmid pWWO of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH : cytochrome c reductase activity. The molecular mass of the eniyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH :acceptor reductase was capable of reducing either one-or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a K , value for NADH of 22 pM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD' (semiquinone fbrm), with a calculated midpoint redox potential of -244 mV. The reduction of FAD' to FAD" (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. 'The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH+FAD+[2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2.6-dichloroindophenol at a reduced rate.Pseudomonas putida containing the TOL plasmid pWWO is capable of utilising aromatic hydrocarbons such as xylenes and toluenc as sole sources of carbon and energy. The TOL plasmid encodes an inducible metabolic pathway permitting the degradation of the hydrocarbons to Krebs cycle intermediates [l]. The genes for this pathway are organised into two operons, the first one encoding three enzymes catalysing the successive oxidation of the methyl side-chain of the aromatic substrates to the carboxylate group [2]. Two of these enzymes, benzyl alcohol dehydrogenasc and benzaldehyde dehydrogenCorrespondence to S . Harayama,

Intracellular proteome expression during 4-n-nonylphenol biodegradation by the filamentous fungus Metarhizium robertsii

“…The criteria for trend analysis was fulfilled by ion 179 m/z, which occurred in all byproducts of 4-n-NP and its intensity was directly related to only one TMS group attached to hydroxyl substituent placed on C1 of the benzene ring. 4-HBAL was chosen as a possible 4-n-NP biodegradation byproduct that occurs in many aerobic pathways for compounds containing a benzene ring with at least one aliphatic carbon substituent (Shaw and Harayama 1992;Spivack et al 1994;Jorgensen et al 1995). Both approaches aided in the formulation of the biodegradation pathway ( fig.…”

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate

“…The monooxygenase is a two-component enzyme consisting of a reductase subunit which transfers electrons from NADH through FAD and a [2Fe2S] center to the membrane-associated hydroxylase subunit. There, one atom of activated molecular oxygen is inserted into the methyl group of the aromatic hydrocarbon substrate while the other oxygen atom is reduced to water (77,81). This enzyme also has activity toward the reaction product, benzyl alcohol (or m-or p-toluic alcohol) (Fig.…”

“…The xyl genes of pWW0 that encode the oxidation of toluene, mxylene, and p-xylene to benzoate, m-toluate, and p-toluate, respectively, have been cloned and sequenced, and some of the enzymes have been purified and studied in detail (39,40,45,(76)(77)(78)81). The pathway is initiated by a monooxygenase (Fig.…”

Isolation and Characterization of a 4-Nitrotoluene-Oxidizing Enzyme from Activated Sludge by a Metagenomic Approach

“…The toluene monooxygenase from Pseudomonas putida TOL-plasmid pWW0 consists of two polypeptide subunits, encoded by xylM and xylA (18,46). XylA, the NADH: acceptor reductase component, is an electron transport protein transferring reducing equivalents from NADH to XylM, the hydroxylase component (42). The toluene monooxygenase (XylMA) expressed in E. coli oxidizes not only toluene and xylene but also m-and p-ethyl-, methoxy-, nitro-, and chloro-substituted toluenes, as well as m-bromo-substituted toluene, to form the corresponding benzyl alcohol derivatives (23).…”