Isolation of a novel thermostable dehydrochlorinase (LinA) from a soil metagenome.
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ABSTRACT:
SUBMITTER: Macwan AS
PROVIDER: S-EPMC3339611 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:LinA is the first enzyme of the microbial degradation pathway of a chlorinated insecticide, hexachlorocyclohexane (HCH), and mediates the dehydrochlorination of ?-, ?-, and ?-HCH. Its two variants, LinA type 1 and LinA type 2, which differ at 10 out of 156 amino acid residues, have been described. Their activities for the metabolism of different HCH isomers differ considerably but overall are high for ?-HCH, moderate for ?-HCH, low for ?-HCH, and lacking for ?-HCH. Here, we describe the characterization of a new variant of this enzyme, LinA type 3, whose gene was identified from the metagenome of an HCH-contaminated soil sample. Its deduced primary structure in the region spanning amino acid residues 1 to 147 of the protein exhibits 17 and 12 differences from LinA type 1 and LinA type 2, respectively. In addition, the residues GIHFAPS, present at the region spanning residues 148 to 154 in both LinA type 1 and LinA type 2, are deleted in LinA type 3.The activity of LinA type 3 for the metabolism of ?-HCH is several orders of magnitude higher than that of LinA type 1 or LinA type 2 and can be useful for improvement of the metabolism of ?-HCH.
| S-EPMC4592873 | biostudies-literature
Project description:Herein we present the results of an in-depth simulation study of LinA and its two variants. In our analysis, we combined the exploration of protein conformational dynamics with and without bound substrates (hexachlorocyclohexane (HCH) isomers) performed using molecular dynamics simulation followed by the extraction of the most frequently visited conformations and their characteristics with a detailed description of the interactions taking place in the active site between the respective HCH molecule and the first shell residues by using symmetry-adapted perturbation theory (SAPT) calculations. A detailed investigation of the conformational space of LinA substates has been accompanied by description of enzymatic catalytic steps carried out using a hybrid quantum mechanics/molecular mechanics (QM/MM) potential along with the computation of the potential of mean force (PMF) to estimate the free energy barriers for the studied transformations: dehydrochlorination of γ-, (-)-α-, and (+)-α-HCH by LinA-type I and -type II variants. The applied combination of computational techniques allowed us not only to characterize two LinA types but also to point to the most important differences between them and link their features to catalytic efficiency each of them possesses toward the respective ligand. More importantly it has been demonstrated that type I protein is more mobile, its active site has a larger volume, and the dehydrochlorination products are stabilized more strongly than in the case of type II enzyme, due to differences in the residues present in the active sites. Additionally, interaction energy calculations revealed very interesting patterns not predicted before but having the potential to be utilized in any attempts of improving LinA catalytic efficiency. On the basis of all these observations, LinA-type I protein seems to be more preorganized for the dehydrochlorination reaction it catalyzes than the type II variant.
| S-EPMC7681783 | biostudies-literature
Project description:Hexachlorocyclohexane dehydrochlorinase (LinA) mediates dehydrochlorination of ?-HCH to 1, 3, 4, 6-tetrachloro-1,4-cyclohexadiene that constitutes first step of the aerobic degradation pathway. We report the 3.5 Å crystal structure of a thermostable LinA-type2 protein, obtained from a soil metagenome, in the hexagonal space group P6(3)22 with unit cell parameters a?=?b?=?162.5, c?=?186.3 Å, respectively. The structure was solved by molecular replacement using the co-ordinates of LinA-type1 that exhibits mesophile-like properties. Structural comparison of LinA-type2 and -type1 proteins suggests that thermostability of LinA-type2 might partly arise due to presence of higher number of ionic interactions, along with 4% increase in the intersubunit buried surface area. Mutational analysis involving the differing residues between the -type1 and -type2 proteins, circular dichroism experiments and functional assays suggest that Q20 and G23 are determinants of stability for LinA-type2. It was earlier reported that LinA-type1 exhibits enantioselectivity for the (-) enantiomer of ?-HCH. Contrastingly, we identified that -type2 protein prefers the (+) enantiomer of ?-HCH. Structural analysis and molecular docking experiments suggest that changed residues K20Q, L96C and A131G, vicinal to the active site are probably responsible for the altered enantioselectivity of LinA-type2. Overall the study has identified features responsible for the thermostability and enantioselectivity of LinA-type2 that can be exploited for the design of variants for specific biotechnological applications.
| S-EPMC3507683 | biostudies-literature
Project description:LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from gamma-hexachlorocyclohexane (gamma-HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via gamma-pentachlorocyclohexene (gamma-PCCH). LinA was crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals belonged to space group P4(1) or P4(3), with unit-cell parameters a = b = 68.9, c = 101.9 A, and diffracted X-rays to 2.25 A resolution. The crystal contained three molecules in the asymmetric unit.
| S-EPMC2720343 | biostudies-literature
Project description:Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.
| S-EPMC5622026 | biostudies-literature
Project description:BackgroundThe alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes.Methodology/principal findingsMetagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C.Conclusion/significanceThis is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues.
| S-EPMC3561394 | biostudies-literature
Project description:The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.
| S-EPMC201203 | biostudies-literature
Project description:The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to beta-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50 degrees C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47 degrees C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.
| S-EPMC1472341 | biostudies-literature
Project description:An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6-9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.
| S-EPMC4009859 | biostudies-literature
Project description:The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable ?-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs of?>500?bp in length. Similarity search for ?-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for ?-xylosidase activity. A total of 82 proteins exhibited ?-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.
| S-EPMC5726482 | biostudies-literature