Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1

Abstract

Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2 , oxidized toluene to p-cresol as the first detectable product. Experiments with p-deuterotoluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 1802, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.Several small aromatic hydrocarbons can be utilized by bacteria as sole carbon and energy sources for growth. Degradation of these hydrocarbons is initiated by either oxidation of an alkyl side group (26,36) or direct attack on the aromatic ring (1,9,11,12,20). Toluene has been used by a number of laboratories as a model compound to study the metabolism of aromatic hydrocarbons. When utilized as a sole carbon and energy source, toluene can be metabolized via oxidation of the methyl group to yield benzyl alcohol as the first metabolite (30,36). This type of oxidation is catalyzed by enzymes of the TOL pathway. Toluene may also be degraded by enzymes of the TOD pathway, in which initial attack is by dioxygenation of the aromatic nucleus at the 2,3 position (11,25,38). In this case, the first detectable product is (+)-cis-l(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). Recently, toluene has been shown to be oxidized by whole cells of organisms which grow on toluene to o-cresol (33) and p-cresol (35a) as initial metabolites.A bacterium which grows on toluene as a sole carbon and energy source has been isolated and identified in our laboratory as Pseudomonas mendocina KR1 (31a). After growth with toluene, whole cells of this organism oxidize p-cresol at a rapid rate. We now report the separation into three protein components of the enzyme system responsible for the conversion of toluene to p-cresol. The reconstituted enzyme system catalyzed oxidation of toluene to p-cresol by an NAD(P)H-dependent monooxygenation reaction in which one atom of molecular oxygen is incorporated into the hydroxyl group of p-cresol. The reaction proceeded with a concomitant NIH shift, which suggests that toluene-3,4-oxide is the first initial oxidation product.( 6,7,8-tetrahydropterine dihydrochloride, DNase I, Bis-Tris propane, Trizma base (Tris), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES), morpholine propane sulfonate (MOPS), dithiothreitol (DTT), acrylamide, and N,N,N',N'-tetramethylethylenediamine were from Sigma Chemical Co., Rockford, Ill.; 2-(N-morpholine)ethanesulfate was from U.S. Biochemical Corp., Cleveland, Ohio; sodium dodecyl sulfate (SDS) was from Fisher Scientific, Fairlawn, N.J.; 2-amino-6,7 -dimethyl -...

Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and p- xylene

“…First, partially purified preparations of T4MO were reported to give Ϸ70% retention of 2 H in p-cresol obtained from 4-2 H 1 -toluene (9). This high percentage of retention indicates that a direct insertion into the COH bond ( Fig.…”

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

“…Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1 is a multicomponent hydroxylase that catalyzes the O 2 and NADH dependent hydroxylation of toluene to form pcresol with high regiospecificity [1,2]. The T4MO complex is encoded by a 4.7 kb gene cluster, tmoA-F [3].…”

“…The T4MO complex is encoded by a 4.7 kb gene cluster, tmoA-F [3]. The six genes in this cluster encode a diiron hydroxylase (T4moH), a cofactorless effector protein (T4moD), a soluble Rieske type ferredoxin (T4moC), and an NADH oxidoreductase (T4moF) [1,3].…”

The enzymatic basis for pesticide bioremediation

“…TOD also acts as a monooxygenase on monocyclic aromatics, aliphatic olefi ns and other miscellaneous substrates [33,34]. By these means it converts different isomers of dimethylbenzene into dimethyl phenols and isomers of nitrotoluene into nitobenzyl alcohols and nitophenols [31,34]. Allylic methyl group monooxygenation can be seen with different halo-propene and halo-butene isomers which are converted into butene-1-ol and propene-1-ol, respectively [35].…”

“…This multi-component enzyme not only has extremely broad substrate specifi city but also acts as a dioxygenase or monooxygenase. TOD acts as a dioxygenase against a range of compounds including monocyclic aromatics, fused aromatics, linked aromatics and aliphatic olefi ns [31,32]. TOD also acts as a monooxygenase on monocyclic aromatics, aliphatic olefi ns and other miscellaneous substrates [33,34].…”