Search Results (118)

Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stress conditions. In the absence of stress, they assemble into large oligomers. In response to stress, such as elevated temperatures, they undergo conformational changes that expose hydrophobic surfaces, allowing them to interact with denatured proteins. At heat shock temperatures in bacteria, large sHsp oligomers disassemble into smaller oligomeric forms. Methanogens are a diverse group of microorganisms, ranging from thermophilic to psychrophilic and halophilic species. Accordingly, their sHsps exhibit markedly different temperature dependencies based on their optimal growth temperatures. In this study, we characterized sHsps from both hyperthermophilic and mesophilic methanogens to investigate the mechanisms underlying their temperature-dependent behavior. Using analytical ultracentrifugation, we observed the dissociation of sHsps from a mesophilic methanogen into dimers. The dissociation equilibrium of these oligomers was found to be dependent not only on temperature but also on protein concentration. Furthermore, by generating various mutants, we identified the specific amino acid residues responsible for the temperature dependency observed. The C-terminal region containing the IXI/V motif and the α-crystallin domain were found to be the primary determinants of oligomer stability and its temperature dependence. Full article

L-asparaginase (L-ASNase) is a key industrial enzyme significant for cancer therapy and the food industry for reducing dietary acrylamide. The hyperthermophilic L-ASNase from Thermococcus sibiricus (TsAI) was previously shown to exhibit high activity and thermostability and is promising for biotechnology. To gain insights into structure-functional relationships of TsAI, determination of the substrate specificity, kinetic parameters, structural characterization, and molecular docking were performed. TsAI characteristics were compared with the TsAI^D54G/T56Q mutant, which exhibited increased activity after a double mutation in the substrate-binding region. TsAI and TsAI^D54G/T56Q were found to display high activity towards D-asparagine—62% and 21% of L-asparaginase activity, respectively—and low L-glutaminase coactivity of ~5%. Restoring the mesophilic-like triad GSQ in the mutant resulted in a two-fold increase in activity towards L-asparagine compared with TsAI. Crystal structures of TsAI forms solved at 1.9 Å resolution revealed that double mesophilic-like mutation increased the flexibility of the loop M51-L57, located in close proximity to the active site. Structural superposition and mutational analysis indicate that mobility of this loop is essential for the activity of thermo-ASNases. Molecular docking, without taking into account the temperature factor, showed that, in contrast to L-asparagine interaction, D-asparagine orientation in the TsAI and TsAI^D54G/T56Q active sites is similar and not optimal for catalysis. Under real conditions, high temperatures can induce structural changes that reduce L-ASNase discrimination towards D-asparagine. Overall, the obtained structural and biochemical data provide a basis for a more detailed understanding of thermo-ASNase functioning and possibilities to engineer improved variants for future biotechnological application. Full article

The Maillard reaction is a network of interconnected interactions yielding in formation a number of toxic derivatives in processed foods. Acrylamide, a potential carcinogen and a product of the Maillard reaction, is formed under food processing, predominantly from asparagine and reducing sugars at temperatures over 120 °C. In this study, we investigated the potency of recombinant hyperthermophilic L-asparaginase from Thermococcus sibiricus TsAI to mitigate dietary acrylamide by hydrolyzing substrate for its synthesis under various operation conditions. Using a simplified food system for self-cooking, high acrylamide levels were found in baked samples regardless of whether L- or D-enantiomer of asparagine was added. TsAI effectively reduced acrylamide content under various pretreatment conditions, such as temperature, concentration, and time of incubation. The lowest acrylamide level of 1.0–1.1% of the control values or 3.52–3.76 µg/kg was observed in samples pretreated with TsAI 20 U/mL at 90 °C for 20–25 min. Due to the exceptionally high D-asparaginase activity of hyperthermophilic TsAI, the dietary acrylamide content formed from D-asparagine was reduced by 54.8% compared to the control. Comparison of the wild-type TsAI and its mutant reveal that an enzyme displaying enhanced stability is more functional for food-processing application. The native TsAI decreased acrylamide level by 98.9%, while the highly active mutant, with increased structural flexibility, decreased it by only 26.8%. TsAI treatment effectively blocked acrylamide synthesis, but not melanoidin formation via the Maillard reaction, thus not affecting sample characteristics such as color (browning) and aroma, which are important for consumer perception. Full article

Archaea thrive in extreme environments, exhibiting unique traits with significant biotechnological potential. In this study, we investigated whether Thermococcus onnurineus NA1 could stably integrate a large glycoside hydrolase (GH) gene cluster from T. pacificus P-4, enhancing β-linked polysaccharides degradation for hydrogen production. Among 35 Thermococcus genomes examined via OrthoFinder2 and OrthoVenn3, and selecting Tpa-GH gene clusters as the target, we cloned and integrated Tpa-GH into T. onnurineus NA1 using a fosmid-based system, creating the GH03 mutant. Cultivation in a modified MM1 medium supplemented with laminarin revealed significantly higher growth and hydrogen production in T. onnurineus GH03 than in the wild-type strain. Our findings demonstrate the feasibility of stable, large-fragment DNA integration in hyperthermophilic archaea and underscore the promise of T. onnurineus GH03 as a strain for high-temperature biomass conversion. Full article

Phosphoglucomutase (EC 5.4.2.2., PGM), a key enzyme of glycogenolysis and glycogenesis, catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate, whereas phosphomannomutase (EC 5.4.2.8., PMM) transfers the phosphate group from the 1′ to the 6′, or from the 6′ to the 1′ position in mannose phosphate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, a single gene, Tk1108, encodes a protein with both PGM and PMM activities. Here, we report biophysical analysis and the 2.45 Å resolution cryo-EM structure of this novel enzyme. Our results demonstrate a specific arrangement of the four subunits in the quaternary structure, displaying a distinct catalytic cleft required for the bifunctional activity at extremely high temperatures. To the best of our knowledge, this is the first biophysical characterization and cryo-EM structure elucidation of a thermostable, bifunctional PGM/PMM. Full article

Extremophiles, microorganisms blooming in extreme environmental conditions, hold particular significance in the domain of microbial research. This review paper focuses on extremophilic microorganisms, emphasizing their adaptations and the diverse products they generate, with a particular emphasis on exopolysaccharides (EPSs). EPSs, high molecular weight carbohydrate biopolymers, stand out as valuable products with applications across various industries. The review explores EPS production by bacteria in extreme conditions, including thermophilic, halophilic, and psychrophilic environments. Noteworthy examples, such as B. thermantarcticus and H. smyrnensis AAD6T, highlight the vast potential of extremophiles in EPS production. Additionally, the paper explores the major synthesis pathways of EPSs, shedding light on the factors influencing biosynthesis. The commercial significance of EPSs, especially for extremophiles, is underlined by their applications in medicine, food, environmental protection, agriculture, cosmetics, and more. Furthermore, the review sheds light on the role of extremophiles in various ecosystems, such as acidophiles, alkaliphiles, halophiles, hyperthermophiles, oligotrophs, osmophiles, piezophiles, and radioresistant organisms. This comprehensive analysis highlights the broad impact of extremophilic microorganisms and their EPS products in scientific exploration and commercial innovation. Full article

Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. Thermococcus kodakarensis, a hyperthermophilic archaeon, harbors a novel enzyme (PPM_Tk) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPM_Tk catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate. Here, we report biophysical analysis, crystallization, and three-dimensional structure determination of PPM_Tk by X-ray diffraction at 2.39 Å resolution. The solved structure revealed a novel catalytic motif, unique to PPM_Tk, which makes this enzyme distinct from the homologous counterparts. We postulate that this novel catalytic motif may enable PPM_Tk to isomerize phosphopentose instead of phosphohexose. To the best of our knowledge, this is the first biophysical and structural analysis of a phosphopentomutase from hyperthermophilic archaea. Full article

DNA polymerases from the hyperthermophilic Archaea have attracted considerable attention as PCR enzymes due to their high thermal stability and proofreading 3′ → 5′ exonuclease activity. This study is the first to report data concerning the purification and biochemical characteristics of the Tst DNA polymerase from Thermococcus stetteri. Both the wild type Tst(wt) DNA polymerase and its chimeric form containing the P36H substitution—which reduces the enzyme’s affinity for the U-containing template and dUTP—and the DNA-binding domain Sso7d from S. solfataricus were obtained and analyzed. It was shown that Tst(wt) could effectively amplify up to 6-kb DNA fragments, whereas TstP36H–Sso7d could amplify DNA fragments up to 15 kb. It was found that TstP36H–Sso7d has superior PCR efficiency compared to the commonly used DNA polymerase PfuV93Q–Sso7d. For the amplification of a 2-kb DNA fragment, TstP36H–Sso7d required less than 10 s of extension time, whereas for PfuV93Q–Sso7d, the extension time was no less than 30 s. Steady-state kinetic assays revealed that the dNTP-binding affinity K^dNTP_m was the same for TstP36H–Sso7d and PfuV93Q–Sso7d, whereas the maximum rate of dNTP incorporation, k_cat, was two orders of magnitude higher for TstP36H–Sso7d. Moreover, the incorporation of incorrect dNTP was not observed for TstP36H–Sso7d up to 56 °C, whereas for PfuV93Q–Sso7d, the extension of primer with incorrect dNTP was observed at 37 °C, supporting higher fidelity of TstP36H–Sso7d. The obtained data suggest that TstP36H–Sso7d may be a good candidate for high-fidelity DNA amplification. Full article

Catalase (CAT) plays a crucial role in plant responses to environmental stresses and maintaining redox homeostasis. However, its putative heat lability might compromise its activity and function, thus restricting plant thermotolerance. Herein, we verified Arabidopsis CAT3 was of poor thermostability that was then engineered by fusion expression in Escherichia coli. We found that our selected fusion partners, three hyperacidic mini-peptides and the short rubredoxin from hyperthermophile Pyrococcus furiosus, were commonly effectual to enhance the solubility and thermostability of CAT3 and enlarge its improvement on heat tolerance in E. coli and yeast. Most importantly, this finding was also achievable in plants. Fusion expression could magnify CAT3-mediated thermotolerance in tobacco. Under heat stress, transgenic lines expressing CAT3 fusions generally outperformed native CAT3 which in turn surpassed wild-type tobacco, in terms of seed germination, seedling survival, plant recovery growth, protection of chlorophyll and membrane lipids, elimination of H₂O₂, as well as mitigation of cell damage in leaves and roots. Moreover, we revealed that the introduced CAT3 or its fusions seemed solely responsible for the enhanced thermotolerance in tobacco. Prospectively, this fusion expression strategy would be applicable to other crucial plant proteins of intrinsic heat instability and thus provide an alternative biotechnological route for ameliorating plant heat tolerance. Full article

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system. Full article

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N⁶-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme–substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature. Full article

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10–50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5–9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90–95 °C to 80–85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use. Full article

Second-generation biofuels from lignocellulosic biomass remain critical and require several challenges due to lignin compounds’ inefficient degradation and recalcitrate characteristics. In this regard, this study focuses on enzymatic technology as a promising treatment that is beneficial in breaking down the biomass’s hemicellulose and cellulosic parts. Thermostable bacterial species owe thermostable enzymes that are able to degrade complex carbohydrate compounds and produce efficient hydrogen production. The present study investigates the direct utilization of ligninolytic enzymes such as cellulase and xylanase derived from the hyperthermophilic bacteria Thermotoga maritima (ATCC 43589 strain). The results show that xylanase and cellulase enzymes extracted from Thermotoga maritima could depolymerize the lignin bonds of corn stover substrate and release monomers such as Galactose in the media. In conclusion, this study can open a new advanced research window on directly applying a hyperthermophilic consortium of enzymes capable of hydrolyzing lignocellulose material toward hydrogen production. Full article

A putative β-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of β-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, K_m, V_max, and K_cat/K_m of BglAc toward pNPG were 357.62 U mg⁻¹, 3.41 mM, 474.0 μmol min⁻¹·mg⁻¹, and 122.7 s⁻¹mM⁻¹. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new β-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation. Full article

Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/β-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs. Full article