Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

Pseudomonas strain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genes atzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous to atzA, -B, and -C. The sequence identities of the atz genes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.

The Atrazine Catabolism Genes atzABC Are Widespread and Highly Conserved

Bacterial dehalogenases catalyse the cleavage of carbon-halogen bonds, which is a key step in aerobic mineralization pathways of many halogenated compounds that occur as environmental pollutants. There is a broad range of dehalogenases, which can be classified in different protein superfamilies and have fundamentally different catalytic mechanisms. Identical dehalogenases have repeatedly been detected in organisms that were isolated at different geographical locations, indicating that only a restricted number of sequences are used for a certain dehalogenation reaction in organohalogen-utilizing organisms. At the same time, massive random sequencing of environmental DNA, and microbial genome sequencing projects have shown that there is a large diversity of dehalogenase sequences that is not employed by known catabolic pathways. The corresponding proteins may have novel functions and selectivities that could be valuable for biotransformations in the future. Apparently, traditional enrichment and metagenome approaches explore different segments of sequence space. This is also observed with alkane hydroxylases, a category of proteins that can be detected on basis of conserved sequence motifs and for which a large number of sequences has been found in isolated bacterial cultures and genomic databases. It is likely that ongoing genetic adaptation, with the recruitment of silent sequences into functional catabolic routes and evolution of substrate range by mutations in structural genes, will further enhance the catabolic potential of bacteria toward synthetic organohalogens and ultimately contribute to cleansing the environment of these toxic and recalcitrant chemicals.

/pdf/bacterial-degradation-of-xenobiotic-compounds-evolution-and-30yz3s06aa.pdf

Bacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities

Microorganisms that can utilize halogenated compounds as a growth substrate generally produce enzymes whose function is carbon-halogen bond cleavage. Based on substrate range, reaction type and gene sequences, the dehalogenating enzymes can be classified in different groups, including hydrolytic dehalogenases, glutathione transferases, monooxygenases and hydratases. X-ray crystallographic and biochemical studies have provided detailed mechanistic insight into the action of haloalkane dehalogenase. The essential features are nucleophilic substitution of the halogen by a carboxylate group and the presence of a distinct halogen binding site, formed by tryptophan residues. This review summaries current knowledge on a variety of other dehalogenating enzymes and indicates the existence of a widespread and diverse microbial potential for dechlorination of natural and xenobiotic halogenated compounds.

Genetics and biochemistry of dehalogenating enzymes

γ-Hexachlorocyclohexane (γ-HCH; also called BHC or lindane) is one of the highly chlorinated pesticides which can cause serious environmental problems. Sphingomonas paucimobilis UT26 degrades γ-HCH under aerobic conditions. The unique degradation pathway of γ-HCH in UT26 is revealed. In the upstream pathway, γ-HCH is transformed to 2,5-dichlorohydroquinone (2,5-DCHQ) by two different dehalogenases (LinA and LinB) and one dehydrogenase (LinC) which are expressed constitutively. In the downstream pathway, 2,5-DCHQ is reductively dehalogenated, and then ring-cleaved by enzymes (LinD and LinE, respectively) whose expressions are regulated. We have cloned and sequenced five structural genes (linA, linB, linC, linD, and linE) directly involved in this degradation pathway. The linD and linE genes form an operon, and its expression is positively regulated by the LysR-type transcriptional regulator (LinR). The genes linA, linB, and linC are constitutively expressed, and are present separately from each other in the UT26 genome. Cell fractionation analysis, Western blotting, and immuno electron microscopy revealed that LinA and LinB are localized in the periplasmic space of UT26.

Complete analysis of genes and enzymes for γ-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26

Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G + C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus. The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.

Lack of homology between two haloacetate dehalogenase genes encoded on a plasmid from Moraxella sp. strain B.

PROBLEM TO BE SOLVED: To reliably prevent the generation of slime such as microorganisms at a drain pan 18 to store drain from an evaporator 7 generated during cooling operation of an air conditioning indoor machine 3. SOLUTION: Water in a storage 22 of a drain pan 18 is drained to an external part by an always driven pump 24, and at other time than a cooling operation time, such as during a stop of cooling operation and heating operation, operation of the pump 24 is rested. During resting of the cooling operation, water present in the pump 24 is reversed in the drain pan 22. In a state that a comparatively high water level 32 is kept, a slime inhibitor 28 is dipped and dissolved and electric conductivity is increased. During a start of cooling operation, if detecting electric conductivity is less than predetermined value, at which the generation of slime can be prevented, this is reported by a reporting means. This shows that the slime inhibitor is consumed out or becomes insufficient and can replenish the slime inhibitor.

Maintenance device for air conditioning indoor machine

PROBLEM TO BE SOLVED: To suppress occurrence of moldy smell and poor drainage and prevent lowering of operation efficiency. SOLUTION: The air conditioner includes a heat exchanger 1 having heat transfer pipes 7, to which at least a cooling refrigerant is supplied, and a drain pan 2 which receives condensate, which is generated on the surface of the exchanger 1 and drips down, to discharge it to the outside. In this case, a fuel cell 50, in which fuel reacts electrochemically with oxygen to generate power, is provided, and a heat cleaning means wherein after the completion of a cooling operation, hot water HW1 discharged from the cell 50 or hot water HW2, which is heated by utilizing waste heat of the fuel cell, is used to heat or clean the surface of the exchanger 1 or a condensate receiving part of the pan 2.

Air conditioner and operation method thereof

PROBLEM TO BE SOLVED: To provide a garbage treating agent and a high-temperature garbage treatment method whereby oil in organic wastes can be efficiently decomposed SOLUTION: There are provided an organic waste treating agent containing oil-decomposing bacteria and an oil adsorbing/adjusting material and a method for treating organic wastes comprising allowing oil-decomposing bacteria to act on oil-containing organic wastes in the presence of an oil adsorbing/adjusting material

Garbage treating agent and high-temperature garbage treatment method

PROBLEM TO BE SOLVED: To efficiently degrade an oil component in an organic waste. SOLUTION: This bacterium belongs to Bacillus stearothermophilus, and is capable of degrading an oil component at 60 deg.C or higher. A method for treating an organic waste using the bacterium, and an apparatus for treating an organic waste are also provided.

Matsushita Isao

Papers

Lack of homology between two haloacetate dehalogenase genes encoded on a plasmid from Moraxella sp. strain B.

Maintenance device for air conditioning indoor machine

Air conditioner and operation method thereof

Garbage treating agent and high-temperature garbage treatment method

Thermophilic oil-degrading bacterium