Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

Biocatalysis in organic solvents(OSs)has numerous important applications,but native enzymes in OSs often exhibit limited catalytic performance.Herein,we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface.Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue(Arg).Particularly,a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance(five-fold improvement in OS resistance and seven-fold half-life increase in 40%(vol)dimethylsulfoxide).As a result,the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA.Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects:(a)improved the overall structural stability,(b)increased the hydrophobicity of the local microenvironment around the catalytic triad,(c)enriched the hydrophobic substrate around the enzyme molecule,and(d)lowered the contact frequency between OS molecules and the catalytic triad.Our findings validate that computationaided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.

Purification and Characterization of 2-Haloacid Dehalogenase from Marine Bacterium Paracoccus sp. DEH99, Isolated from Marine Sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.

Interaction Between 1,2,3-Trichloropropane and Haloalkane Dehalogenase LinB

The haloalkane dehalogenase LinB from Sphingomonas paucimobills UT26 was found to transform the 1,2,3-trichloropropane（TCP） into inorganic halide ions and 2,3-dichloro-1-propanol although the catalytic activity is very low（Kcat=0.005 s^-1）.In this study,molecular dynamics simulation and docking studies were performed to investigate the binding of TCP to LinB.The docking results indicate that LinB does not restrict TCP to be bound productively in the active site and the water-mediated inhibition occurs in the process of TCP interacting with LinB.The residues Ile134,Leu150,Phe154,Pro208,and Ile211 located on the cap domain are potential targets for mutagenesis researches.

Characteristics of dehalogenase from bacteria isolated from the Gut of Pond-reared Rohu (<i>Labeo rohita</i>) Juveniles in Myanmar

Unwarranted accumulation of halogenated compounds in the rivers and streams has in recent years emerged due to the widespread use agricultural pesticides. The presence of these halogenated compounds in the water does not only suppress the immune system of fish but adversely induce serious morbidity and mortality among cultured stocks. Importantly, gradual accumulation of these compounds in the system of cultured and wild freshwater fish species cultured in ponds and floating net-cages in dams and rivers, respectively, poses some risks to humans, the end users. In this study, we attempted to isolate bacteria from the gut of pond-reared rohu (Labeo rohita) in Myanmar, screened the isolated bacteria for dehalogenase gene using molecular technique and tested the ability of these bacteria to degrade halogenated compounds in vitro. The eight bacterial strains studied were identified as Enterobacter mori strain MK- 121001, Enterobacter cloacae strains MK121003, MK-121004, MK121010, Ralstonia solanacearum strain 121002, Acinetobacter baumannii strain MK121007, Chromobacterium violaceum strain MK121009 and Pantoea vagans strain 121011. Only three bacterial strains (MK121002, MK121007 and MK121009) were capable of degrading 2,2-dichloropropionic acid (2,2-DCP) as the sole carbon source up to a final substrate concentration of 20 mM. Their mean growth doubling time ranging from 6-23 hours with the maximum of chloride ion released of 85%. PCR amplifica- tion with oligonucleotide primers designed from group I dehalogenase revealed the presence of deha- logenase genes in all isolates suggesting dehalogenase gene in strains 121001, 121003, 121004, 121010 and 121011 were silenced. In contrast, group II dehalogenase primers did not show any PCR amplification. These results suggest that MK121002, MK121007 and MK121009 only encode a group I dehalogenase and its non-stereoselectivity is in agreement with previoulsly described group I haloacid dehalogenase. The partial gene sequences were blasted but no significant sequence identity was observed. Therefore, it suggest the 2-haloacid dehalogenase of MK121002, MK12-1007 and MK121009 might be a novel group I 2-haloacid dehalogenase. The results indicated a broad distribution of dehalogenation genes in many micro- bial genomes that harbor dehalogenase(s) due to the exposure of the microorganisms to the naturally occurring or man-made halogenated compounds in the environmental systems. So far, microorganisms capable of producing dehalogenases were mainly isolated from soil and scarcely from aquatic animals and their environments. To the authors’ knowledge, this is the first report on the isolation of dehalogenase-producing bacteria from the gut of pond-reared freshwater fish, Labeo rohita, in Myanmar.

Structure-guided protein design of fluoroacetate dehalogenase for kinetic resolution of rac-2-bromobutyric acid

R-2-Bromobutyric acid is a very important intermediate for the synthesis of agrochemicals and pharmaceuticals.Bioresolution of rac-2-bromobutyric acid(rac-2-BBA)provides a promising process for R-2-bromobutyric acid(R-2-BBA)production.The fluoroacetate dehalogenase(FAcD)has been always studied in the defluorination process.We found that FAcD RPA1163 showed detectable activity but no enantioselectivity towards rac-2-BBA.The iterative saturation mutagenesis(ISM)of FAcD RPA1163 resulted in a mutant H155V/W156R/Y219M,which catalyzed the kinetic resolution of rac-2-BBA to produce R-2-BBA with enhanced activity and enantioselectivity(99.3%ee).The high preference for S-2-bromobutyric acid(S-2-BBA)is of synthetic value.Molecular docking analysis indicated that the H155V/W156R/Y219M mutation reduced steric hindrance and broadened the halide pocket.It is not only the steric hindrance but also the electrostatic environment that has an effect on the activity and enantioselectivity.

Aerobic dechlorination of cis- and trans-dichloroethenes by some indigenous bacteria isolated from contaminated sites in Africa

The innate toxicity of dichloroethenes(DCEs) and their tendency to be reduced to vinyl chloride(VC)(a known human carcinogen) is a cause for environmental concern. Aerobic bacteria capable of growth on cis- and trans-DCEs as sole carbon and energy sources were isolated by enrichment culture technique and identified to belong to the genera; Bacillus, Pseudomonas and Acinetobacter. Axenic and mixed cultures of the bacterial isolates utilized DCEs at concentrations above the maximum contaminant level allowable in drinking water by the Environmental Protection Agency. Their specific growth rate constant ranged significantly(P<0.05) between 0.346—0.552 and 0.461—0.667 d -1; while the maximum specific substrate utilization rate ranged significantly(P<0.05) between 20.01—29.79 and 31.40—42.83 nmol h -1 (mg of protein) -1 in cis- and trans-DCE, respectively. The optimum growth was observed at 30℃ and at a pH of 7.0 with up to 96% of the stoichiometric-expected chloride released. Serial adaptation positively affected the growth yields and dehalogenase activities of the organisms with multiple antibiotic patterns also demonstrated by the isolates. These findings therefore indicated the important roles that these organisms may play in the bioremediation of sites polluted with chlorinated ethene compounds in Africa.

Fluoroacetate in plants - a review of its distribution, toxicity to livestock and microbial detoxification

Fluoroacetate producing plants grow worldwide and it is believed they produce this toxic compound as a defence mechanism against grazing by herbivores. Ingestion by livestock often results in fatal poisonings, which causes significant economic problems to commercial farmers in many countries such as Australia, Brazil and South Africa.Several approaches have been adopted to protect livestock from the toxicity with limited success including fencing,toxic plant eradication and agents that bind the toxin. Genetically modified bacteria capable of degrading fluoroacetate have been able to protect ruminants from fluoroacetate toxicity under experimental conditions but concerns over the release of these microbes into the environment have prevented the application of this technology.Recently, a native bacterium from an Australian bovine rumen was isolated which can degrade fluoroacetate. This bacterium, strain MFA1, which belongs to the Synergistetes phylum degrades fluoroacetate to fluoride ions and acetate. The discovery and isolation of this bacterium provides a new opportunity to detoxify fluoroacetate in the rumen. This review focuses on fluoroacetate toxicity in ruminant livestock, the mechanism of fluoroacetate toxicity,tolerance of some animals to fluoroaceate, previous attempts to mitigate toxicity, aerobic and anaerobic microbial degradation of fluoroacetate, and future directions to overcome fluoroacetate toxicity.

Evaluation of Transgenic <i>Nicotiana tabacum</i>with <i>deh</i>E Gene Using Transposon Based IRAP Markers

In the present study, five genetically modified herbicide tolerant Nicotiana tabacum cv. TAPM24 plants with a constructed vector pCAMBIA1301a carrying dehalogenase E (dehE) gene were compared with three non-transgenic controls using Tto1 retrotransposon specific IRAP markers. dehE gene was originally characterized in Rhizobium sp. and it produced an enzyme which degraded the Dalapon herbicide. IRAP protocol was applied on transgenic and non-transgenic plants to investigate the retrotransposon based genetic variation which may appear during transformation. Polymorphism rates were calculated as 0%-20% from IRAP-PCR products among all plant samples. These results show that transformation of tobacco plant with the dehE gene may cause Tto1 retrotransposon alterations appearing as different band profiles. The findings are expected to contribute to genetic engineering studies to obtain better results and also to understand how transposons contribute to features such as transgenesis. In our knowledge, this is one of the first experimental data of transgenic N. tabacum engineered with dehE gene originated Rhizobium sp. in terms of retrotranposon based variation.

Individual stages of bacterial dichloromethane degradation mapped by carbon and chlorine stable isotope analysis

The fractionation of carbon and chlorine stable isotopes of dichloromethane(CH_2Cl_2,DCM)upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed.Isotope effects for individual steps of the multi-stage DCM degradation process,including transfer across the cell wall from the aqueous medium to the cell cytoplasm,dehalogenase binding,and catalytic reaction,were considered.The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of C\Cl bonds,and not by inflow of DCM into cells.Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures,presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess.Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes(Δδ^(13)C/Δδ^(37)Cl),increasing corresponding ratio from 7.2–8.6 to 9.6–10.5 under conditions of glutathione deficiency.This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM,unlike the uncatalyzed reaction,which is a one-stage process,as shown by quantum-chemical modeling.