Toluene Dioxygenase Research Articles (Page 5)

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

Chemoenzymatic total syntheses of the linear triquinane-type natural products (+)-hirsutic acid and (−)-complicatic acid from toluene

Diasterodivergent synthesis of optically pure vinyl episulfides and β-hydroxy thiocyanates from a bacterial metabolite

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylation of aromatic compounds. To achieve high efficient biotransformation of benzene to benzene cis-diols, Pseudomonas putida KT2442, Pseudomonas stutzeri 1317, and Aeromonas hydrophila 4AK4 were used as hosts to express TDO gene tod. Plasmid pSPM01, a derivative of broad-host plasmid pBBR1MCS-2 harboring tod from plasmid pKST11, was constructed and introduced into the above three strains. Their abilities to catalyze the biotransformation of benzene to benzene cis-diols, namely, cis-3,5-cyclohexadien-1,2-diols abbreviated as DHCD, were examined. In shake-flask cultivation under optimized culture media and growth condition, benzene cis-diols production by recombinant P. putida KT2442 (pSPM01), P. stutzeri 1317 (pSPM01), and A. hydrophila 4AK4 (pSPM01) were 2.68, 2.13, and 1.17 g/l, respectively. In comparison, Escherichia coli JM109 (pSPM01) and E. coli JM109 (pKST11) produced 0.45 and 0.53 g/l of DHCD, respectively. When biotransformation was run in a 6-l fermenter, DHCD production in P. putida KT2442 (pSPM01) was approximately 60 g/l; this is the highest DHCD production yield reported so far.

Cupriavidus necator (formerly Ralstonia eutropha) strain H850 is known to grow on biphenyl, and to co-oxidize congeners of polychlorinated biphenyls (PCBs). Using a Tn5-based minitransposon shuttle system and the TOL plasmid, the rational construction of hybrids of H850 was achieved by subsequent introduction of three distinct elements carrying 11 catabolic loci from three other biodegrading bacteria into the parent strain, finally yielding C. necator RW112. The new genetic elements introduced into H850 and its derivatives were tcbRCDEF, which encode the catabolic enzymes needed for chlorocatechol biodegradation under the control of a transcriptional regulator, followed by cbdABC, encoding a 2-halobenzoate dioxygenase, and xylXYZ, encoding a broad-spectrum toluate dioxygenase. The expression of the introduced genes was demonstrated by measuring the corresponding enzymic activities. The engineered strain RW112 gained the ability to grow on all isomeric monochlorobenzoates and 3,5-dichlorobenzoate, all monochlorobiphenyls, and 3,5-dichloro-, 2,3'-dichloro- and 2,4'-dichlorobiphenyl, without accumulation of chlorobenzoates. It also grew and utilized two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as sole carbon and energy sources for growth. This is the first report on the aerobic growth of a genetically improved bacterial strain at the expense of technical Aroclor mixtures.

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Toluene dioxygenase-mediated oxidation of dibromobenzenes. Absolute stereochemistry of new metabolites and synthesis of (−)-conduritol E

The TodS and TodT proteins form a previously unrecognized and highly specific two-component regulatory system in which the TodS sensor protein contains two input domains, each of which are coupled to a histidine kinase domain. This system regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. In contrast to the narrow substrate range of this catabolic pathway, the TodS effector profile is broad. TodS has basal autophosphorylation activity in vitro, which is enhanced by the presence of effectors. Toluene binds to TodS with high affinity (Kd = 684 +/- 13 nM) and 1:1 stoichiometry. The analysis of the truncated variants of TodS reveals that toluene binds to the N-terminal input domain (Kd = 2.3 +/- 0.1 microM) but not to the C-terminal half. TodS transphosphorylates TodT, which binds to two highly similar DNA binding sites at base pairs -107 and -85 of the promoter. Integration host factor (IHF) plays a crucial role in the activation process and binds between the upstream TodT boxes and the -10 hexamer region. In an IHF-deficient background, expression from the tod promoter drops 8-fold. In vitro transcription assays confirmed the role determined in vivo for TodS, TodT, and IHF. A functional model is presented in which IHF favors the contact between the TodT activator, bound further upstream, and the alpha-subunit of RNA polymerase bound to the downstream promoter element. Once these contacts are established, the tod operon is efficiently transcribed.

Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

The kinetics of toluene degradation as a function of oxygen concentration were compared for six strains of toluene-oxidizing bacteria using initial rate assays. The effect of nitrate was also examined. Rates of degradation and the relative effect of oxygen on the degradation rate were correlated with the pathway for toluene oxidation. Strains which synthesize toluene dioxygenases, Pseudomonas putida F1, P. fluorescens CFS215, and Pseudomonas sp. strain W31, degraded toluene at significantly higher rates (151–166 nmol/mg per min) than strains synthesizing toluene monooxygenases, Burkholderia cepacia G4 (23 nmol/mg per min) and B. pickettii PKO1 (14 nmol/mg per min), or a methylmonooxygenase, P. putida PaW1 (12 nmol/mg per min). Rates declined 30–48% for the dioxygenase strains and 25% for PaW1 as the oxygen concentration was decreased from 240 to 50 μM, but declined less than 10% for G4 and PKO1. Nitrate enhanced toluene degradation by the denitrifying strains PKO1 and W31 at oxygen concentrations below 30 μM, but had no significant effect on any of the other strains. Biphasic kinetics were observed for all of the strains, with double-reciprocal plots of the data exhibiting an inflection point at a ‘critical oxygen concentration’ between 20 and 30 μM. Below this concentration, the rate of toluene degradation was inhibited to a greater extent than predicted by the kinetic data for higher oxygen concentrations. For the denitrifying strains PKO1 and W31, however, monophasic kinetics were observed when nitrate was provided as an alternative electron acceptor. These observations suggest that biphasic kinetics result when rates of toluene degradation are limited by the availability of electron acceptor at the critical oxygen concentration, and that this limitation is overcome by denitrifying strains able to respire nitrate. Taken together, our findings suggest that the synthesis of monooxygenases and the ability to denitrify represent independent adaptations for toluene utilization in low oxygen environments. Moreover, these data support the use of nitrate in mixed electron acceptor strategies for the bioremediation of contaminated aquifers, as well as the targeted use of monooxygenase and dioxygenase strains in settings in which their physiological traits can be best exploited.

A series of 2-, 3- and 4-substituted pyridines was metabolised using the mutant soil bacterium Pseudomonas putida UV4 which contains a toluene dioxygenase (TDO) enzyme. The regioselectivity of the biotransformation in each case was determined by the position of the substituent. 4-Alkylpyridines were hydroxylated exclusively on the ring to give the corresponding 4-substituted 3-hydroxypyridines, while 3-alkylpyridines were hydroxylated stereoselectively on C-1 of the alkyl group with no evidence of ring hydroxylation. 2-Alkylpyridines gave both ring and side-chain hydroxylation products. Choro- and bromo-substituted pyridines, and pyridine itself, while being poor substrates for P. putida UV4, were converted to some extent to the corresponding 3-hydroxypyridines. These unoptimised biotransformations are rare examples of the direct enzyme-catalysed oxidation of pyridine rings and provide a novel synthetic method for the preparation of substituted pyridinols. Evidence for the involvement of the same TDO enzyme in both ring and side-chain hydroxylation pathways was obtained using a recombinant strain of Escherichia coli (pKST11) containing a cloned gene for TDO. The observed stereoselectivity of the side-chain hydroxylation process in P. putida UV4 was complicated by the action of an alcohol dehydrogenase enzyme in the organism which slowly leads to epimerisation of the initial (R)-alcohol bioproducts by dehydrogenation to the corresponding ketones followed by stereoselective reduction to the (S)-alcohols.

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric substituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations.

Enhancement of cometabolic biodegradation of trichloroethylene (TCE) gas in biofiltration

Processing of cyclopropylarenes by toluene dioxygenase: isolation and absolute configuration of metabolites

Biocatalytic synthesis of monocyclic arene-dihydrodiols and -diols by Escherichia coli cells expressing hybrid toluene/biphenyl dioxygenase and dihydrodiol dehydrogenase genes

Kinetic analyses of trichloroethylene cometabolism by toluene-degrading bacteria harboring a tod homologous gene

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.

Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme system consisting of a reductase (ReductaseTOL), a ferredoxin (FerredoxinTOL) and a Rieske non-heme iron dioxygenase (OxygenaseTOL). The three components and the apoenzyme of the dioxygenase (apo-OxygenaseTOL) were overexpressed, purified and crystallized. ReductaseTOL diffracts to 1.8 A and belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 77.1, c = 156.3 A. Ferredoxin(TOL) diffracts to 1.2 A and belongs to space group P2(1), with unit-cell parameters a = 30.5, b = 52.0, c = 30.95 A, beta = 113.7 degrees. Apo-OxygenaseTOL and OxygenaseTOL diffract to 3.2 A and belong to space group P4(3)32, with unit-cell parameters a = 235.9 A and a = 234.5 A, respectively.

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.

Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, alpha and beta; the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the alpha subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase alpha subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the alpha subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.