Biochemical Characterization of a Novel Haloalkane Dehalogenase from a Cold-Adapted Bacterium

Drienovská, Ivana; Chovancová, Eva; Koudeláková, Táňa; Damborský, Jiřı́; Chaloupková, Radka

doi:10.1128/aem.00485-12

Cited by 34 publications

(33 citation statements)

References 38 publications

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“…It has very high specific activity with 1-bromohexane and 1,3-dibromopropane, but also has significant activity with 1,5-dichloropropane (132 nmol s activity with chlorinated compounds, unlike DppA, DpcA, and DmrA. [17][18][19] The K m values for DccA are on par with reported values, whereas the k cat values are up to 10-fold higher than reported for other HLD-I subfamily members (Table I). Our structure of DccA adds to our structural understanding of the HLD-I enzymes, with structures for DmrA, DppA, and DhlA having been previously reported.…”

Section: Specific Activity and Kinetic Parameterssupporting

confidence: 42%

“…HLD-I subfamily members DpcA from Psychrobacter cryohalolentis, DmrA from Mycobacterium rhodesiae strain JS60, and DppA from Plesiocystis pacifica SIR-1 all showed (Table I). [17][18][19] This substrate specificity profile is characteristic of substrate specificity group (SSG)-IV; by contrast, DhlA is categorized in SSG-I. Both DmbB from Mycobacterium bovis 18 and DhmA from Mycobacterium avium N85 21 were categorized in the HLD-I subfamily but their substrate specificities were not reported due to expression and stability problems.…”

Section: Introductionmentioning

confidence: 99%

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Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

et al. 2016

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Two putative haloalkane dehalogenases (HLDs) of the HLD-I subfamily, DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea, have been identified based on sequence comparisons with functionally characterized HLD enzymes. The two genes were synthesized, functionally expressed in E. coli and shown to have activity toward a panel of haloalkane substrates. DsaA has a moderate activity level and a preference for long (greater than 3 carbons) brominated substrates, but little activity toward chlorinated alkanes. DccA shows high activity with both long brominated and chlorinated alkanes. The structure of DccA was determined by X-ray crystallography and was refined to 1.5 Å resolution. The enzyme has a large and open binding pocket with two well-defined access tunnels. A structural alignment of HLD-I subfamily members suggests a possible basis for substrate specificity is due to access tunnel size.

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Section: Specific Activity and Kinetic Parameterssupporting

confidence: 42%

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

et al. 2016

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“…Figure S9). 45 (C) Michaelis−Menten plots for 10 haloalkane substrates tested with DbjA with the fits superimposed. The kinetics parameters can be extracted with high accuracy (SI, Table 1).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Interfacing Microwells with Nanoliter Compartments: A Sampler Generating High-Resolution Concentration Gradients for Quantitative Biochemical Analyses in Droplets

et al. 2014

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Analysis of concentration dependencies is key to the quantitative understanding of biological and chemical systems. In experimental tests involving concentration gradients such as inhibitor library screening, the number of data points and the ratio between the stock volume and the volume required in each test determine the quality and efficiency of the information gained. Titerplate assays are currently the most widely used format, even though they require microlitre volumes. Compartmentalization of reactions in pico- to nanoliter water-in-oil droplets in microfluidic devices provides a solution for massive volume reduction. This work addresses the challenge of producing microfluidic-based concentration gradients in a way that every droplet represents one unique reagent combination. We present a simple microcapillary technique able to generate such series of monodisperse water-in-oil droplets (with a frequency of up to 10 Hz) from a sample presented in an open well (e.g., a titerplate). Time-dependent variation of the well content results in microdroplets that represent time capsules of the composition of the source well. By preserving the spatial encoding of the droplets in tubing, each reactor is assigned an accurate concentration value. We used this approach to record kinetic time courses of the haloalkane dehalogenase DbjA and analyzed 150 combinations of enzyme/substrate/inhibitor in less than 5 min, resulting in conclusive Michaelis-Menten and inhibition curves. Avoiding chips and merely requiring two pumps, a magnetic plate with a stirrer, tubing, and a pipet tip, this easy-to-use device rivals the output of much more expensive liquid handling systems using a fraction (∼100-fold less) of the reagents consumed in microwell format.

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“…Over the past decade, a variety of thermophilic (Rye et al 2009), psychrophilic (Drienovska et al 2012;Novak et al 2013), and non-extremophilic microbes capable of degrading 2,4-D (Fulthorpe et al 1995;González et al 2012;Kumar et al 2014;Samir et al 2015) have been isolated from contaminated marine environments (Chiba et al 2009;Fulthorpe et al 1995;González et al 2012;Kumar et al 2014;Novak et al 2014;Samir et al 2015). However, studies on bioremediation of chlorinated compounds under hypersaline environments are sparse.…”

Section: Degradation Of Halogenated Hydrocarbons By Halophilic Bactermentioning

confidence: 97%

Halophiles: biology, adaptation, and their role in decontamination of hypersaline environments

Edbeib

Wahab

Huyop

2016

World J Microbiol Biotechnol

138

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The unique cellular enzymatic machinery of halophilic microbes allows them to thrive in extreme saline environments. That these microorganisms can prosper in hypersaline environments has been correlated with the elevated acidic amino acid content in their proteins, which increase the negative protein surface potential. Because these microorganisms effectively use hydrocarbons as their sole carbon and energy sources, they may prove to be valuable bioremediation agents for the treatment of saline effluents and hypersaline waters contaminated with toxic compounds that are resistant to degradation. This review highlights the various strategies adopted by halophiles to compensate for their saline surroundings and includes descriptions of recent studies that have used these microorganisms for bioremediation of environments contaminated by petroleum hydrocarbons. The known halotolerant dehalogenase-producing microbes, their dehalogenation mechanisms, and how their proteins are stabilized is also reviewed. In view of their robustness in saline environments, efforts to document their full potential regarding remediation of contaminated hypersaline ecosystems merits further exploration.

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Biochemical Characterization of a Novel Haloalkane Dehalogenase from a Cold-Adapted Bacterium

Cited by 34 publications

References 38 publications

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

Interfacing Microwells with Nanoliter Compartments: A Sampler Generating High-Resolution Concentration Gradients for Quantitative Biochemical Analyses in Droplets

Halophiles: biology, adaptation, and their role in decontamination of hypersaline environments

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