Sequence analysis of the gene cluster encoding toluene-3-monooxygenase from Pseudomonas pickettii PKO1

Byrne, Armando M.; Kukor, Jerome J.; Olsen, Ronald H.

doi:10.1016/0378-1119(94)00844-i

Cited by 97 publications

(99 citation statements)

References 24 publications

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“…The V106A mutation lies in helix B (based on the sMMO crystal structure [11,32]) of the hydroxylase ␣-subunit. Interestingly, this amino acid is always a hydrophobic amino acid in all other related nonheme monooxygenases (1,4,5,7,19,28,46). TOM-Green, however, is the first monooxygenase to have an alanine at this position, just three amino acids away from a conserved glutamate involved in binding the diiron catalytic center of the hydroxylase where oxidation occurs.…”

Section: Resultsmentioning

confidence: 99%

Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

et al. 2002

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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 ␣-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.

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Section: Resultsmentioning

confidence: 99%

Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

et al. 2002

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“…Dichloromethane was distilled over calcium hydride and diethyl ether over Na wire. Organic solutions were dried over anhydrous MgSO 4 and, unless stated otherwise, were evaporated at 14 mm Hg. Thin-layer chromatography was performed by using Aldrich Silica Gel 60 plates (F254).…”

Section: Methodsmentioning

confidence: 99%

Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

Zhou¹,

et al. 2002

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The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3-and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the "classical" naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.The classical pathway for naphthalene catabolism in bacteria such as Pseudomonas putida strains NCIMB9816 and PpG7 (7, 52, 53) is via dihydroxylation and cleavage of the first ring and removal of the resulting aliphatic side chain to produce salicylate (2-hydroxybenzoate). This is then converted by the action of salicylate 1-hydroxylase to catechol (1,2-dihydroxybenzene), which undergoes extradiol cleavage via the same route used for a wide range of other aromatic compounds such as toluene and the xylenes (16) and phenol (38). The nah genes are located on two separate operons: the upper pathway operon encoding the conversion of naphthalene to salicylate and the lower (or meta) pathway operon encoding the conversion of salicylate to acetyl coenzyme A and pyruvate (5, 52). The induction of the two operons is linked by a common regulator protein, NahR (34).Although a route for naphthalene catabolism involving the alternative conversion of salicylate to gentisate (2,5-dihydroxybenzoate) has been known for some time (14,25,30,39,41,53), genetic analysis of such a pathway, in Ralstonia (formerly Pseudomonas) sp. strain U2, has only recently been reported (12, 54). There is similarity between the nag genes of strain U2 and the classical nah genes, but only in the conversion of naphthalene to salicylate, for which the genes are homologous and in the same ord...

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“…strain M (McDonald et al, 1997), Pseudomonas sp. strain CF600 (Nordlund et al, 1990), Pseudomonas putida strain H (Herrmann et al, 1995), Pseudomonas putida F1 , Pseudomonas mendocina (Yen et al, 1991), Burkholderia cepacia G4 (Shields et al, 1989), Ralstonia pickettii (objective synonym of Pseudomonas pickettii) (Byrne et al, 1995) and Ralstonia eutropha (objective synonym of Alcaligenes eutrophus) (Kim et al, 1996). …”

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confidence: 99%

Burkholderia kururiensis sp. nov., a trichloroethylene (TCE)-degrading bacterium isolated from an aquifer polluted with TCE.

Zhang¹,

Hanada²,

Shigematsu³

et al. 2000

International Journal of Systematic and Evolutionary Microbiology

122

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A trichloroethylene (TCE)-degrading bacterium was isolated from an aquifer sample collected at a TCE-polluted site in Japan by enriching with phenol as sole carbon source. The isolate, designated strain KP23 T , was a Gram-negative, oval-shaped micro-organism. A phylogenetic study based on 16S rRNA gene sequences indicated that strain KP23 T should be placed in the genus Burkholderia. Cellular fatty acids of the strain were mainly composed of C 16 :0 , cyclopropanic acid C 17 :0 and cyclopropanic acid C 19 :0 . Strain KP23 T also contained notable amounts of C 13 :1 and C 17 :1 . The GMC content of total DNA was 64 8 mol %. Strain KP23 T oxidized various sugars and sugar alcohols as sole carbon source such as galactose, glucose, mannose, maltose, glycerol, inositol and mannitol. Comparisons of its phenotypic and genotypic characteristics with other known species belonging to the genus Burkholderia suggested that strain KP23 T represents a new species in the genus. The name Burkholderia kururiensis is proposed for this species, with strain KP23 T as the type strain (l JCM 10599 T ).Keywords : trichloroethylene degradation, Burkholderia kururiensis sp. nov., aquifer, 16S rRNA gene INTRODUCTIONTrichloroethylene (TCE) has been used as a solvent in manufacturing to clean grease from machinery all over the world. Like many other chlorinated hydrocarbons, it has become one of the most abundant environmental pollutants because of its toxic properties and widespread occurrence in soil and groundwater in many countries. TCE removal by dumping or air stripping is restricted by legislation and thus attention is now being paid to biological degradation in soil and groundwater. In the past decade, research on the application of bacteria to the aerobic biodegradation of TCE has progressed and a wide variety of microorganisms has been investigated for this purpose. Most of these bacteria belong to the Proteobacteria such as Methylococcus capsulatus (Stainthorpe et al., 1990) Abbreviations : FAME, fatty acid methyl ester ; TCE, trichloroethylene ; UQ, ubiquinone.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KP23 T is AB024310. OB3b (Cardy et al., 1991), Methylocystis sp. strain M (McDonald et al., 1997), Pseudomonas sp. strain CF600 (Nordlund et al., 1990), Pseudomonas putida strain H (Herrmann et al., 1995), Pseudomonas putida F1 , Pseudomonas mendocina (Yen et al., 1991), Burkholderia cepacia G4 (Shields et al., 1989), Ralstonia pickettii (objective synonym of Pseudomonas pickettii) (Byrne et al., 1995) and Ralstonia eutropha (objective synonym of Alcaligenes eutrophus) (Kim et al., 1996). Methylosinus trichosporiumIn these organisms, TCE degradation is catalysed by monoxygenases or dioxygenases which are induced by specific substrates relevant to the enzymes. The inducers include various aromatic and aliphatic hydrocarbons such as toluene (Nelson et al., 1987), phenol (Folsom et al., 1990Harker & Kim, 1990), isopropylbenzene (Dabrock et al., 1992), propane and methane (Little et al., 1988 ;...

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Sequence analysis of the gene cluster encoding toluene-3-monooxygenase from Pseudomonas pickettii PKO1

Cited by 97 publications

References 24 publications

Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

Burkholderia kururiensis sp. nov., a trichloroethylene (TCE)-degrading bacterium isolated from an aquifer polluted with TCE.

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