Reaction participants Show >> << Hide
- Name help_outline H+ Identifier CHEBI:15378 Charge 1 Formula H InChIKeyhelp_outline GPRLSGONYQIRFK-UHFFFAOYSA-N SMILEShelp_outline [H+] 2D coordinates Mol file for the small molecule Search links Involved in 9,176 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline NADH Identifier CHEBI:57945 (Beilstein: 3869564) help_outline Charge -2 Formula C21H27N7O14P2 InChIKeyhelp_outline BOPGDPNILDQYTO-NNYOXOHSSA-L SMILEShelp_outline NC(=O)C1=CN(C=CC1)[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OC[C@H]2O[C@H]([C@H](O)[C@@H]2O)n2cnc3c(N)ncnc23)[C@@H](O)[C@H]1O 2D coordinates Mol file for the small molecule Search links Involved in 1,073 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline O2 Identifier CHEBI:15379 (CAS: 7782-44-7) help_outline Charge 0 Formula O2 InChIKeyhelp_outline MYMOFIZGZYHOMD-UHFFFAOYSA-N SMILEShelp_outline O=O 2D coordinates Mol file for the small molecule Search links Involved in 2,648 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline toluene Identifier CHEBI:17578 (Beilstein: 635760; CAS: 108-88-3) help_outline Charge 0 Formula C7H8 InChIKeyhelp_outline YXFVVABEGXRONW-UHFFFAOYSA-N SMILEShelp_outline Cc1ccccc1 2D coordinates Mol file for the small molecule Search links Involved in 6 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline 2-hydroxytoluene Identifier CHEBI:28054 (Beilstein: 506917; CAS: 95-48-7) help_outline Charge 0 Formula C7H8O InChIKeyhelp_outline QWVGKYWNOKOFNN-UHFFFAOYSA-N SMILEShelp_outline Cc1ccccc1O 2D coordinates Mol file for the small molecule Search links Involved in 3 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline H2O Identifier CHEBI:15377 (Beilstein: 3587155; CAS: 7732-18-5) help_outline Charge 0 Formula H2O InChIKeyhelp_outline XLYOFNOQVPJJNP-UHFFFAOYSA-N SMILEShelp_outline [H]O[H] 2D coordinates Mol file for the small molecule Search links Involved in 6,048 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline NAD+ Identifier CHEBI:57540 (Beilstein: 3868403) help_outline Charge -1 Formula C21H26N7O14P2 InChIKeyhelp_outline BAWFJGJZGIEFAR-NNYOXOHSSA-M SMILEShelp_outline NC(=O)c1ccc[n+](c1)[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OC[C@H]2O[C@H]([C@H](O)[C@@H]2O)n2cnc3c(N)ncnc23)[C@@H](O)[C@H]1O 2D coordinates Mol file for the small molecule Search links Involved in 1,142 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Cross-references
RHEA:20349 | RHEA:20350 | RHEA:20351 | RHEA:20352 | |
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Reaction direction help_outline | undefined | left-to-right | right-to-left | bidirectional |
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Publications
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Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation.
Canada K.A., Iwashita S., Shim H., Wood T.K.
Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and ... >> More
Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 +/- 0.3 versus 1.39 +/-0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 +/- 0.03 versus 0.029 +/-0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 +/-0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the alpha-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry. << Less
J Bacteriol 184:344-349(2002) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes.
Yeager C.M., Bottomley P.J., Arp D.J., Hyman M.R.
High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inh ... >> More
High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min-1 to 2.45 min-1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne-ethylene and propylene-were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 microM and 112.3 nmol min-1 mg of protein-1 for ethylene and 32.3 microM and 89.2 nmol min-1 mg of protein-1 for propylene. << Less
Appl Environ Microbiol 65:632-639(1999) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.
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Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4.
Newman L.M., Wackett L.P.
Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to b ... >> More
Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity. << Less
Biochemistry 34:14066-14076(1995) [PubMed] [EuropePMC]
This publication is cited by 1 other entry.