Specificity and Kinetics of Haloalkane Dehalogenase

Schanstra, Joost P.; Kingma, Jaap; Janssen, Dick B.

doi:10.1074/jbc.271.25.14747

Cited by 99 publications

(157 citation statements)

References 20 publications

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“…The latter dehalogenase exhibits the highest activity on smaller compounds such as its natural substrate 1,2-dichloroethane and the brominated analogue and nematocide 1,2-dibromoethane (20). Interestingly, DhaA converted 1,2-dibromoethane at a steady-state rate that is 4-5-fold higher than that found with DhlA, even though 1,2-dichloroethane is not a substrate for DhaA (20,25,26).…”

Section: Resultsmentioning

confidence: 94%

“…Furthermore, DhaA converted different brominated propanes with a similar k cat value. The higher affinity of DhaA for brominated compounds might be a consequence of tight substrate binding in combination with a high rate of carbon-bromine bond cleavage, as was observed for 1,2-dibromoethane conversion by DhlA, the corresponding enzyme from X. autotrophicus GJ10 (20). DhaA dehalogenated mono-and dibrominated propanes with a catalytic efficiency similar to that of DhlA, whereas the catalytic efficiency of DhaA on 1,2,3-tribromopropane was ∼30-fold higher (25,26).…”

Section: Resultsmentioning

confidence: 98%

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Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

et al. 2003

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 98%

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

et al. 2003

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“…Samples were taken at different times (0 to 60 min), and absorbance was measured at 460 nm after the addition of mercuric thiocyanate and ferric ammonium sulfate (39). One unit of enzyme activity is defined as the amount of the enzyme that catalyzes the formation of 1 mol of a halide per min.…”

Section: Methodsmentioning

confidence: 99%

A Pseudomonas putida Strain Genetically Engineered for 1,2,3-Trichloropropane Bioremediation

Samin

Pavlová

Arif

et al. 2014

Appl Environ Microbiol

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c 1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCPdegrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.

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“…In structural terms, they belong to the ␣/␤-hydrolase superfamily (2-4). The HLDs have broad substrate specificity, enabling them to catalyze conversion of diverse chlorinated, brominated and iodinated alkanes, alkenes, cycloalkanes, alcohols, epoxides, carboxylic acids, esters, ethers, amides, and nitriles (5,6).…”

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confidence: 99%

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Buryška

Daniel

Kunka

et al. 2016

Appl Environ Microbiol

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bHaloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 M and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization. Haloalkane dehalogenases (HLDs; EC 3.5.1.8) are enzymes that catalyze the hydrolytic cleavage of carbon-halogen bonds in alkyl halides ( Fig. 1) with a wide range of potential applications in biocatalysis, biodegradation, biosensing, decontamination, and cell imaging (1). In structural terms, they belong to the ␣/␤-hydrolase superfamily (2-4). The HLDs have broad substrate specificity, enabling them to catalyze conversion of diverse chlorinated, brominated and iodinated alkanes, alkenes, cycloalkanes, alcohols, epoxides, carboxylic acids, esters, ethers, amides, and nitriles (5, 6).The first known members of this family were isolated from the bacteria Xanthobacter autotrophicus GJ10, Sphinghomonas paucimobilis UT26, and Rhodococcus rhodochrous NCIMB13064 (3, 7, 8), which colonize environments that have been heavily contaminated with halogenated pollutants, such as 1,2-dichloroethane, 1,2,3,4,5,6-hexachlorocyclohexane, and 1-chlorobutane. In these microorganisms, the HLDs were found to be components of metabolic pathways that enable the microbes to utilize otherwise toxic haloalkanes as their sole source of carbon and energy. The HLDs have been recently discovered in a wider range of organisms, including symbiotic bacteria such as Bradyrhizobium japonicum USDA110 and Mesorhizobium loti MAF303099 (9, 10), pathogenic bacteria such as Agrobacterium tumefaciens C58 and Mycobacterium spp. (11,12), and the eukaryotic organism Strongylocentrotus purpuratus (13). Even though HLDs have been studied intensively over the last 25 years and isolated from many different environments and species, most of their biological functions remain elusive. For instance, it is undoubtedly interesting that the plant pathogen Agrobacterium tumefaciens C58 carries the HLDencoding gene datA on its tumor-inducing plasmid while there is no clear link between HLD activity and tumorigenesis (1...

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Specificity and Kinetics of Haloalkane Dehalogenase

Cited by 99 publications

References 20 publications

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

A Pseudomonas putida Strain Genetically Engineered for 1,2,3-Trichloropropane Bioremediation

Discovery of Novel Haloalkane Dehalogenase Inhibitors

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