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Alpha-subunit positions methionine 180 and glutamate 214 of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence catalysis.

ABSTRACT: Alpha-subunit position M180 of toluene-o-xylene monooxygenase influences the regiospecific oxidation of aromatics (e.g., from o-cresol, M180H forms 3-methylcatechol, methylhydroquinone, and 4-methylresorcinol, whereas the wild type forms only 3-methylcatechol). Position E214 influences the rate of reaction (e.g., E214G increases p-nitrophenol oxidation 15-fold) by controlling substrate entrance and product efflux as a gate residue.

SUBMITTER: Vardar G

PROVIDER: S-EPMC545643 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Alpha-subunit positions methionine 180 and glutamate 214 of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence catalysis.

Vardar Gönül G Wood Thomas K TK

Journal of bacteriology 20050201 4

Alpha-subunit position M180 of toluene-o-xylene monooxygenase influences the regiospecific oxidation of aromatics (e.g., from o-cresol, M180H forms 3-methylcatechol, methylhydroquinone, and 4-methylresorcinol, whereas the wild type forms only 3-methylcatechol). Position E214 influences the rate of reaction (e.g., E214G increases p-nitrophenol oxidation 15-fold) by controlling substrate entrance and product efflux as a gate residue. ...[more]

PMID: 15687216

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Project description:Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.

Dataset Information

Alpha-subunit positions methionine 180 and glutamate 214 of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence catalysis.

Publications

Alpha-subunit positions methionine 180 and glutamate 214 of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence catalysis.

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