Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para -Hydroxylating Enzyme

Abstract

Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ؎ 0.33 nmol/min/mg of protein with an apparent K m value of 250 M and produced 90% p-cresol and 10% m-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p-cresol and 3% m-cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1 H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.Monooxygenases catalyze the introduction of an oxygen atom into a substrate while the second oxygen atom is reduced to water with electrons from NAD(P)H. This complex reaction often requires a metal center, and electron transfer from the reduced cofactors is mediated by additional proteins (15,17). Oxidizing enzymes are of great potential to the chemical and pharmaceutical industries due to their high regio-, chemo-, and stereoselectivity and their ability to facilitate reactions with chemically stable substrates (1, 2). Due to their complexity, biological oxidation reactions are often performed using growing or resting cells (17).One important group of bacterial oxygenases includes the toluene monooxygenases, which have additional potential applications in bioremediation (23,30,32). The aerobic biodegradation of toluene is well studied, and the pathway is available at the University of Minnesota databank (9). Xylene monooxygenase of Pseudomonas putida mt-2 hydroxylates toluene at the methyl side chain, resulting in benzyl alcohol (1). Toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates the benzene ring at the ortho position to form ocresol, which is further oxidized to 3-methylcatechol (20, 29). Toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported to hydroxylate...

Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone

“…Kinetic analysis of o-, m-, and p-cresol oxidation by wild-type T4MO showed that this enzyme follows typical saturation kinetics with these three cresol substrates; the apparent V max and K m values were 7.6 Ϯ 2.2 nmol/min/mg of protein and 0.13 Ϯ 0.05 mM, respectively, for 3-methylcatechol formation from o-cresol, 10.7 Ϯ 0.1 nmol/min/mg of protein and 0.18 Ϯ 0.01 mM, respectively, for 4-methylcatechol formation from m-cresol, and 6.6 Ϯ 0.9 nmol/min/mg of protein and 0.18 Ϯ 0.02 mM, respectively, for 4-methylcatechol formation from p-cresol. The rates of methylcatechol formation were similar to the oxidation rate of the physiological substrate, toluene (the apparent V max of toluene oxidation for wild-type T4MO was 15.1 Ϯ 0.8 nmol/min/mg of protein [10]); hence, the rates of methylcatechol formation are significant.…”

“…T4MO of P. mendocina KR1 is the most efficient tolueneoxidizing enzyme in the toluene monooxygenase family, which includes TOM (26), toluene para-monooxygenase (formerly toluene 3-monooxygenase [10]) of Ralstonia pickettii PKO1, and toluene/o-xylene monooxygenase of Pseudomonas stutzeri OX1 (4). T4MO oxidizes toluene to 96% p-cresol, 2.8% mcresol, 0.4% o-cresol, and 0.8% benzyl alcohol (29).…”

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

“…Metabolic degradation is less common, with only a few microorganisms being able to use dioxane as the sole source for carbon and energy (18,(30)(31)(32). Cometabolic pathways include several monooxygenase enzymes required for dioxane degradation, including the methane (MMO), propane (PrMO), phenol (PHE), tetrahydrofuran (THFMO), and toluene (TOL, T4MO, or RMO), monooxygenases (19,20,28,33,34). THFMO was proven to catalyze THF as well as dioxane biodegradation in Pseudonocardia sp.…”

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway

“…Initially, toluene monooxygenase of R. pickettii PKO1 was reported to hydroxylate toluene at the meta position, producing primarily 3-methylphenol (61). However, later studies have shown that this monooxygenase hydroxylates toluene predominantly at the para position producing 4-methylphenol (62). The different temporal and absolute occurrences of the tmo, phe, and exdo-aet-14 genes indicate the presence of microbes with different gene inventories than AET-6-14.…”