The minimal structure for iodotyrosine deiodinase function is defined by an outlier protein from the thermophilic bacterium Thermotoga neapolitana.

The nitroreductase superfamily of enzymes encompasses many flavin mononucleotide (FMN)-dependent catalysts promoting a wide range of reactions. All share a common core consisting of an FMN-binding domain, and individual subgroups additionally contain one to three sequence extensions radiating from defined positions within this core to support their unique catalytic properties. To identify the minimum structure required for activity in the iodotyrosine deiodinase subgroup of this superfamily, attention was directed to a representative from the thermophilic organism Thermotoga neapolitana (TnIYD). This representative was selected based on its status as an outlier of the subgroup arising from its deficiency in certain standard motifs evident in all homologues from mesophiles. We found that TnIYD lacked a typical N-terminal sequence and one of its two characteristic sequence extensions, neither of which was found to be necessary for activity. We also show that TnIYD efficiently promotes dehalogenation of iodo-, bromo-, and chlorotyrosine, analogous to related deiodinases (IYDs) from humans and other mesophiles. In addition, 2-iodophenol is a weak substrate for TnIYD as it was for all other IYDs characterized to date. Consistent with enzymes from thermophilic organisms, we observed that TnIYD adopts a compact fold and low surface area compared with IYDs from mesophilic organisms. The insights gained from our investigations on TnIYD demonstrate the advantages of focusing on sequences that diverge from conventional standards to uncover the minimum essentials for activity. We conclude that TnIYD now represents a superior starting structure for future efforts to engineer a stable dehalogenase targeting halophenols of environmental concern.

Kinetic modeling of hydrogen and L-lactic acid production by Thermotoga neapolitana via capnophilic lactic fermentation of starch.

This study investigated the feasibility of hydrogen (H2) and L-lactic acid production from starch under capnophilic lactic fermentation (CLF) conditions by using Thermotoga neapolitana. Batch experiments were performed in 120 mL serum bottles and a 3 L pH-controlled continuous stirred-tank reactors (CSTR) system with potato and wheat starch as the substrates. A H2 yield of 3.34 (±0.17) and 2.79 (±0.17) mol H2/mol of glucose eq. was achieved with, respectively, potato and wheat starch. In the presence of CO2, L-lactic acid production by the acetyl-CoA carboxylation was significantly higher for the potato starch (0.88 ± 0.39 mol lactic acid/mol glucose eq.) than wheat starch (0.33 ± 0.11 mol lactic acid/mol glucose eq.). A kinetic model was applied to simulate and predict the T. neapolitana metabolic profile and bioreactor performance under CLF conditions. The CLF-based starch fermentation suggests a new direction to biotransform agri-food waste into biofuels and valuable biochemicals.

3D reconstruction and morphological analysis of electrostimulated hyperthermophile biofilms of Thermotoga neapolitana.

In this study, the morphological characteristics of the T. neapolitana biofilms on a ceramic carrier, stainless steel, graphite foil, carbon paper, carbon felt and carbon cloth using 3D reconstruction technology was investigated. This was based on the micrographs available in Squadrito et al. (Data Brief 33: 106-403, 2020). Besides the ceramic carrier, the other surfaces were conductive and slightly positively polarised (0.8 and 1.2 V). A simple drying technique was used to show the biofilm and avoid its detachment while chemical fixing with glutaraldehyde was used to better highlight the bacterial morphology within the biofilm. The latter was more suitable for investigating biofilm morphology while the former for bacteria morphology. For the ceramic carrier and stainless steel electrode surfaces, a regular undulating pattern of the biofilm was highlighted by the 3D rendering whilst the glutaraldehyde fixed sample showed a rod-like bacteria morphology. For the other surfaces, a regular undulating pattern of the biofilm and a mixture of a rod-like and a coccoid form of settled bacteria were evidenced also. Carbon cloth was the more suitable electrode for the current application due to its richer filamentous network of bacteria biofilm suggesting a better prevention of bacteria detachment from the electrode surface. Indeed, a preserved biofilm was highlighted on the surfaces of the polarised carbon cloth.

A novel bifunctional aldehyde/alcohol dehydrogenase catalyzing reduction of acetyl-CoA to ethanol at temperatures up to 95 °C.

Hyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80-85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Electrostimulation of hyperthermophile Thermotoga neapolitana cultures.

Hyperthermophile bioelectrochemical systems are seldom investigated although their superior control of microbial consortium and thermodynamic advantages. Hyperthermophilic Thermotogales, for instance, are able to produce hydrogen and lactic acid from wastes better than mesophilic bacteria. Here, the electrostimulation of Thermotoga neapolitana in single-chamber electrochemical bioreactors is studied. The glucose fermentation under CO2 pressure, as model metabolism, was tested at 80 °C. Results show that a dynamic polarization (±0.8 to ±1.2 V) drives glucose fermentation and biofilm stasis on electrodes. Under this condition, production of lactic acid (33 vs 12 mM) and yields of acetate and hydrogen (with lactic/acetic acid ratio of 1.18) were higher than those achieved with static polarization or open-circuit. Dynamic polarization is possibly exploitable to stimulate T. neapolitana in a hyperthermophile electrochemical system for various applications including control of power-to-gas processes or production of value-added products (hydrogen and lactic acid) from sugary wastes.

Efficient xylan-to-sugar biotransformation using an engineered xylanase in hyperthermic environment.

Hyperthermophilic xylanases play a critical role in bioconversion from xylan to sugar in the process of biomass utilization. The discovery of new or improvement of existing xylanases based on directed evolution is expected to be an effective approach to meet the increasing demand of thermostable xylanases. In this work, a xylanase B gene (CTN_0623) from Thermotoga neapolitana (Tne) was cloned and xylanase B (herein named TnexlnB) was solubly expressed in E. coli with a high amount using a heat shock vector pHsh. TnexlnB showed the highest endo-ß-1,4-xylan hydrolase activity at 75 °C and pH 6.0. Additionally, 1 mM Mg2+, Ba2+ and Ca2+ improved the activity of TnexlnB by 31%, 37%, and 53%, respectively. The optimal temperature reached 85 °C by site-directed mutation at the last three helices of TnexlnB. Km and Vmax towards birchwood xylan were determined for both wide type and the best mutant, as follow: 1.09 mg/mL, 191.76 U/mg and 0.29 mg/mL, 376.42 U/mg, respectively. Further characterization highlighted good thermal stability (>80% of enzymatic activity after 1 h at 90 °C) for the best mutant, which made this enzyme suitable for biomass degradation at high temperature.

Biochemical characterisation of four rhamnosidases from thermophilic bacteria of the genera Thermotoga, Caldicellulosiruptor and Thermoclostridium.

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm_Ram106B, Tn_Ram106B, Cb_Ram106B and Ts_Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-L-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as Km, Vmax and kcat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC50) between 5 mM and 8 mM. The α-L-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.

Improvement in activity of cellulase Cel12A of Thermotoga neapolitana by error prone PCR.

Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7-, 5- and 4.8-fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported.

High rate continuous biohydrogen production by hyperthermophilic Thermotoga neapolitana.

This study focused on continuous-flow hydrogen production by Thermotoga neapolitana at a hydraulic retention time (HRT) decreasing from 24 to 5 h. At each HRT reduction, the hydrogen yield (HY) immediately dropped, but recovered during prolonged cultivation at constant HRT. The final HY in each operating period decreased from 3.4 (±0.1) to 2.0 (±0.0) mol H2/mol glucose when reducing the HRT from 24 to 7 h. Simultaneously, the hydrogen production rate (HPR) and the liquid phase hydrogen concentration (H2aq) increased from 82 (±1) to 192 (±4) mL/L/h and from 9.1 (±0.3) to 15.6 (±0.7) mL/L, respectively. Additionally, the effluent glucose concentration increased from 2.1 (±0.1) to above 10 mM. Recirculating H2-rich biogas prevented the supersaturation of H2aq reaching a value of 9.3 (±0.7) mL/L, resulting in complete glucose consumption and the highest HPR of 277 mL/L/h at an HRT of 5 h.

Enzymatic transformation of ginsenosides Re, Rg1, and Rf to ginsenosides Rg2 and aglycon PPT by using ß-glucosidase from Thermotoga neapolitana.

OBJECTIVES: To enzymatically transform protopanaxatriol by using ß-glucosidase from Thermotoga neapolitana (T. neapolitana) DSM 4359. RESULTS: Recombinant ß-glucosidase was purified, which molecular weight was about 79.5 kDa. High levels of ginsenoside were obtained using the follow reaction conditions: 2 mg ml-1 ginsenoside, 25 U ml-1 enzyme, 85 °C, and pH 5.0. ß-glucosidase converted ginsenoside Re to Rg2, Rf and Rg1 to APPT completely after 3 h under the given conditions, respectively. The enzyme created 1.66 mg ml-1 Rg2 from Re with 553 mg l-1 h-1, 0.85 mg ml-1, and 1.01 mg ml-1 APPT from Rg1 and Rf with 283 and 316 mg l-1 h-1 APPT. CONCLUSIONS: ß-glucosidase could be useful for the high-yield, rapid, and low-cost preparation of ginsenoside Rg2 from Re, and APPT from the ginsenosides Rg1 and Rf.

Design of a highly thermostable hemicellulose-degrading blend from Thermotoga neapolitana for the treatment of lignocellulosic biomass.

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-ß-galactanase and an α-l-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (kcat values of ˜177 and ˜133 s-1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases - endo-1,4-ß-xylanase (XynA), endo-1,4-ß-mannanase (ManB/Man5A) and ß-glucosidase (GghA) - to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.

Effect of feed glucose and acetic acid on continuous biohydrogen production by Thermotoga neapolitana.

This study focused on the effect of feed glucose and acetic acid on biohydrogen production by Thermotoga neapolitana under continuous-flow conditions. Increasing the feed glucose concentration from 11.1 to 41.6 mM decreased the hydrogen yield from 3.6 (±0.1) to 1.4 (±0.1) mol H2/mol glucose. The hydrogen production rate concomitantly increased until 27.8 mM of feed glucose but remained unaffected when feed glucose was further raised to 41.6 mM. Increasing the acetic acid concentration from 0 to 240 mM hampered dark fermentation in batch bioassays, diminishing the cumulative hydrogen production by 45% and the hydrogen production rate by 57%, but induced no negative effect during continuous operation. Indeed, throughout the continuous flow operation the process performance improved considerably, as indicated by the 47% increase of hydrogen yield up to 3.1 (±0.1) mol H2/mol glucose on day 110 at 27.8 mM feed glucose.

Identifying conformational changes with site-directed spin labeling reveals that the GTPase domain of HydF is a molecular switch.

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases.

Crystal structure of ß-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides.

The ß-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal ß-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4' and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-ß-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872-884. © 2016 Wiley Periodicals, Inc.

Rational design of a thermostable glycoside hydrolase from family 3 introduces ß-glycosynthase activity.

The thermostable ß-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric three-domain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a ß-glucosynthase, where three nucleophilic variants were created (TnBgl3B_D242G, TnBgl3B_D242A, TnBgl3B_D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into ß-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an α-glucosyl fluoride donor with a 4-nitrophenyl-ß-d-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a ß-1,3-linked disaccharide product, while a secondary ß-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%.

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 ß-glucosidase.

Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 ß-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (r s/r h) from 0.33 to 1.45-2.71. Further increase in r s/r h was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5-10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.

Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories.

The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, ß-1,4-mannotriose, and ß-1,4-mannotetraose. The CelE orthologs additionally bind ß-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind ß-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts.

Model development and experimental validation of capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana.

The aim of the present study was to develop a kinetic model for a recently proposed unique and novel metabolic process called capnophilic (CO2-requiring) lactic fermentation (CLF) pathway in Thermotoga neapolitana. The model was based on Monod kinetics and the mathematical expressions were developed to enable the simulation of biomass growth, substrate consumption and product formation. The calibrated kinetic parameters such as maximum specific uptake rate (k), semi-saturation constant (kS), biomass yield coefficient (Y) and endogenous decay rate (kd) were 1.30 h(-1), 1.42 g/L, 0.1195 and 0.0205 h(-1), respectively. A high correlation (>0.98) was obtained between the experimental data and model predictions for both model validation and cross validation processes. An increase of the lactate production in the range of 40-80% was obtained through CLF pathway compared to the classic dark fermentation model. The proposed kinetic model is the first mechanistically based model for the CLF pathway. This model provides useful information to improve the knowledge about how acetate and CO2 are recycled back by Thermotoga neapolitana to produce lactate without compromising the overall hydrogen yield.

Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU-10.

A 1.3 kb xyl-A gene encoding xylose isomerase from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10 (TnapXI) was cloned and over-expressed in Escherichia coli to produce the enzyme in mesophilic conditions that work at high temperature. The enzyme was concentrated by lyophilization and purified by heat treatment, fractional precipitation, and UNOsphere Q anion-exchange column chromatography to homogeneity level. The apparent molecular mass was estimated by SDS-PAGE to be 49.5 kDa. The active enzyme showed a clear zone on Native-PAGE when stained with 2, 3, 5-triphenyltetrazolium chloride. The optimum temperature and pH for D-glucose to D-fructose isomerization were 98 °C and 7.0, respectively. Xylose isomerase retains 85% of its activity at 50 °C (t1/2 1732 min) for 4 h and 32.5% at 90 °C (t1/2 58 min) for 2 h. It retains 90-95% of its activity at pH 6.5-7.5 for 30 min. The enzyme was highly activated (350%) with the addition of 0.5 mM Co(2+) and to a lesser extent about 180 and 80% with the addition of 5 and 10 mM Mn(2+) and Mg(2+) , respectively but it was inhibited (54-90%) in the presence of 0.5-10 mM Ca(2+) with respect to apo-enzyme. D-glucose isomerization product was also analyzed by Thin Layer Chromatography (Rf 0.65). The enzyme was very stable at neutral pH and sufficiently high temperature and required only a trace amount of Co(2+) for its optimal activity and stability. Overall, 52.2% conversion of D-glucose to D-fructose was achieved by TnapXI. Thus, it has a great potential for industrial applications.