Dataset Information

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

ABSTRACT: Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.

SUBMITTER: Cafaro V

PROVIDER: S-EPMC383105 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

Cafaro Valeria V Izzo Viviana V Scognamiglio Roberta R Notomista Eugenio E Capasso Paola P Casbarra Annarita A Pucci Piero P Di Donato Alberto A

Applied and environmental microbiology 20040401 4

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for m ...[more]

PMID: 15066815

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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.

Project description:Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.

Dataset Information

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

Publications

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes.

Similar Datasets

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