Evidence for Identical Dichloromethane Dehalogenases in Different Methylotrophic Bacteria

Kohler-Staub, D.; Hartmans, S.; Gälli, René; Suter, Franz; Leisinger, Thomas

doi:10.1099/00221287-132-10-2837

Cited by 28 publications

(37 citation statements)

References 14 publications

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“…An open reading frame with a coding capacity of 287 amino acids (molecular weight, 37,430) was identified as demA by its agreement with the N-terminal amino acid sequence, the total amino acid composition, and the subunit size of the purified enzyme. Alignment of the deduced dichloromethane dehalogenase amino acid sequence with amino acid sequences of the functionally related eucaryotic glutathione S-transferases revealed three regions containing highly conserved amino acid residues and indicated that dcmA is a member of the glutathione (16,27). The first step in the degradation of this xenobiotic compound is catalyzed by DCM dehalogenase.…”

mentioning

confidence: 99%

Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family

Leisinger¹

1990

J Bacteriol

158

116

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The nucleotide sequence of a cloned 2.8-kilobase-pair BamHI-PstI fragment containing dcmA, the dichloromethane dehalogenase structural gene from Methylobacterium sp. strain DM4, was determined. An open reading frame with a coding capacity of 287 amino acids (molecular weight, 37,430) was identified as demA by its agreement with the N-terminal amino acid sequence, the total amino acid composition, and the subunit size of the purified enzyme. Alignment of the deduced dichloromethane dehalogenase amino acid sequence with amino acid sequences of the functionally related eucaryotic glutathione S-transferases revealed three regions containing highly conserved amino acid residues and indicated that dcmA is a member of the glutathione (16,27). The first step in the degradation of this xenobiotic compound is catalyzed by DCM dehalogenase. This glutathione (GSH)-dependent enzyme dechlorinates DCM to formaldehyde and inorganic chloride (31). DCM dehalogenase has a hexameric structure with a subunit molecular weight of approximately 33,000. Its catalytic activity is low, but the enzyme is strongly inducible by DCM. Upon growth on DCM, 15 to 20% of the total soluble protein consist of DCM dehalogenase. The purified enzymes of four different facultative methylotrophic bacteria, including Methylobacterium sp. strain DM4, have been shown to be catalytically and immunologically similar and to have identical N-terminal amino acid sequences and subunit molecular weights (16). These observations suggested that the genes for DCM utilization were horizontally distributed among facultative methylotrophic bacteria. The DCM utilization genes of Methylobacterium sp. strain DM4 were isolated by complementation of the DCM-nonutilizing (DCM-) mutant strain DM4-2cr with a clone of a cosmid library of DM4 wild-type DNA in the broad-host-range vector pVK100 (10). It was also demonstrated that the genes for DCM utilization are located on the chromosome or on a megaplasmid and that none of the three cryptic plasmids carried by strain DM4 is involved in DCM metabolism (10).The evolutionary origin of DCM dehalogenase and its potential relatedness to known bacterial enzymes are a matter of speculation. To explore these questions and to provide a basis for studies on the expression of the enzyme, we determined the nucleotide sequence of the dcmA gene, the structural gene of DCM dehalogenase, and its flanking regions. We found that this enzyme had regions of amino * Corresponding author. acid sequences that were conserved in eucaryotic GSH S-transferases and that it exhibited the same percentage of amino acid sequence similarity to eucaryotic GSH S-transferases as the eucaryotic GSH S-transferases among themselves. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The bacteria and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown at 37°C in nutrient yeast broth (NYB) or on nutrient agar (30) containing the appropriate antibiotics at the following concentrations: ampicillin, 100 to 200 jig/...

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confidence: 99%

Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family

Leisinger¹

1990

J Bacteriol

158

116

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“…(20). In all of the aerobic methylotrophs tested, the catalytic activity of DCM dehalogenase is low and the enzyme is 50-to 80-fold induced by DCM (18,26). The genes responsible for DCM utilization have been examined in Methylobacterium sp.…”

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confidence: 99%

“…Aerobic methylotrophic bacteria utilizing dichloromethane (DCM) as the sole carbon and energy source possess the enzyme DCM dehalogenase (18,26). This enzyme converts DCM to formaldehyde and inorganic chloride in a glutathione-dependent reaction (20).…”

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confidence: 99%

Identification of dcmR, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4

Leisinger¹

1991

J Bacteriol

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The genes for dichloromethane utilization by Methylobacterium sp. strain DM4 are encoded on a 2.8-kb sequenced DNA fragment, the dcm region. This fragment contains dcmA, the structural gene of dichloromethane dehalogenase and, upstream of dcmA, a 1. (20). In all of the aerobic methylotrophs tested, the catalytic activity of DCM dehalogenase is low and the enzyme is 50-to 80-fold induced by DCM (18,26). The genes responsible for DCM utilization have been examined in Methylobacterium sp. strain DM4, a pinkpigmented facultative methylotrophic bacterium. They are encoded on a 2.8-kb BamHI-PstI DNA fragment located on the chromosome or on an undetected megaplasmid of this organism (19), and three cryptic plasmids carried by strain DM4 are unrelated to DCM metabolism (10). A 0.9-kb segment of the cloned and sequenced 2.8-kb BamHI-PstI fragment encodes dcmA, the structural gene of DCM dehalogenase. dcmA has been shown to belong to the glutathione S-transferase supergene family. DCM dehalogenase expression is subject to negative control at the transcriptional level, and our preliminary observations suggested that a regulatory protein responsible for this control is also encoded on the sequenced fragment, in the upstream region of dcmA (19).In this communication, we report the functional analysis of the 1.5-kb region upstream of dcmA. We present evidence that this region contains dcmR, the structural gene of a trans-active protein controlling dcmA expression as well as its own synthesis.

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“…The dehalogenase of the bacterium described here, strain GJ70, is more active with secondary substituted bromoalkanes, and also showed activity with brominated alcohols, chlorinated ethers and dihalomethanes, compounds which have not previously been shown to be substrates for hydrolytic enzymes. Dibromomethane and bromochloromethane are also substrates for the dichloromethane S-transferase described by Kohler-Staub et al [13] in dichloromethane-utilizing organisms, but this enzyme acts by a different mechanism. Dichloromethane was not hydrolyzed by the enzyme from strain GJ70.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers

Janssen

Gerritse

Brackman

et al. 1988

European Journal of Biochemistry

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An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6-dichlorohexane-degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28000. The affinity for its substrates was relatively low with K , values for short-chain haloalkanes in the range 0.1 -0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5 -C9 1 -halo-n-alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e. g. methylbromide, dibromomethane, 1,2-dibromoethane, 1,3-dichloropropene, and bis(2-chloroethy1)ether. The degradation of chiral2-bromoalkanes appeared to proceed without stereochemical preference. Optically active 2-bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.Biodegradation of chlorinated organic compounds is dependent on the presence of biochemical activities that can labilize or cleave carbon-halogen bonds. In this respect, an efficient class of enzymes are the bacterial dehalogenases or halidohydrolases. These enzymes are capable of hydrolytic cleavage of carbon-halogen bonds by catalyzing nucleophilic substitution with water. Several dehalogenases with activity towards short-chain 2-halocarboxylic acids have been obtained since 1971 from a variety of organisms that are able to use such compounds as sole carbon source for growth Until recently, it was assumed that in both prokaryotic and eukaryotic organisms, oxidative or reductive reactions, catalyzed by cytochrome P450 or other monooxygenases, or nucleophilic displacement reactions, catalyzed by glutathione transferase, predominate in the biological dehalogenation and detoxification of chlorinated hydrocarbons that do not contain other functional groups such as carboxylic functions [2, 41. Although there is considerable information on the degradation of chlorinated hydrocarbons in liver tissue, hydrolytic dehalogenations have not been demonstrated to be involved in the initial metabolic step [4].We have recently discovered, however, that several chlorinated hydrocarbons of environmental and toxicological relevance may be converted by hydrolytic dehalogenation in bacteria [5 -71. A hydrolytic dehalogenase with activity toward compounds such as 1,2-dichloroethane, 1,2-dibromoethane, 3-chloropropene and methylchloride was described in a strain of Xanthobacter autotrophicus that utilizes chlorinated aliphatics for growth [5, 71. This dehalogenase was found to be a polypeptide with M , 36000 showing activity toward various C1 -C4 1-halogenated or m,o-dihalogenated n-al-[l-31.

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Evidence for Identical Dichloromethane Dehalogenases in Different Methylotrophic Bacteria

Cited by 28 publications

References 14 publications

Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family

Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family

Identification of dcmR, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers

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