Search

Electron bifurcation (BF) is an evolutionarily ancient energy coupling mechanism in anaerobes, whose associated enzymatic machinery remains enigmatic. In BF-flavoenzymes, a chemically high-potential electron forms in a thermodynamically favorable fashion by simultaneously dropping the potential of a second electron before its donation to physiological acceptors. The cryo-EM and spectroscopic analyses of the BF-enzyme Fix/EtfABCX from Thermotoga maritima suggest that the BF-site contains a special flavin-adenine dinucleotide and, upon its reduction with NADH, a low-potential electron transfers to ferredoxin and a high-potential electron reduces menaquinone. The transfer of energy from high-energy intermediates must be carefully orchestrated conformationally to avoid equilibration. Herein, anaerobic size exclusion-coupled small-angle X-ray scattering (SEC-SAXS) shows that the Fix/EtfAB heterodimer subcomplex, which houses BF- and electron transfer (ET)-flavins, exists in a conformational equilibrium of compacted and extended states between flavin-binding domains, the abundance of which is impacted by reduction and NAD(H) binding. The conformations identify dynamics associated with the T. maritima enzyme and also recapitulate states identified in static structures of homologous BF-flavoenzymes. Reduction of Fix/EtfABCX's flavins alone is insufficient to elicit domain movements conducive to ET but requires a structural "trigger" induced by NAD(H) binding. Models show that Fix/EtfABCX's superdimer exists in a combination of states with respect to its BF-subcomplexes, suggesting a cooperative mechanism between supermonomers for optimizing catalysis. The correlation of conformational states with pathway steps suggests a structural means with which Fix/EtfABCX may progress through its catalytic cycle. Collectively, these observations provide a structural framework for tracing Fix/EtfABCX's catalysis.

Electron bifurcation is an energy-conservation mechanism in which a single enzyme couples an exergonic reaction with an endergonic one. Heterotetrameric EtfABCX drives the reduction of low-potential ferredoxin (E° ∼ -450 mV) by oxidation of the midpotential NADH (E° = -320 mV) by simultaneously coupling the reaction to reduction of the high-potential menaquinone (E° = -74 mV). Electron bifurcation occurs at the NADH-oxidizing bifurcating-flavin adenine dinucleotide (BF-FAD) in EtfA, which has extremely crossed half-potentials and passes the first, high-potential electron to an electron-transferring FAD and via two iron-sulfur clusters eventually to menaquinone. The low-potential electron on the BF-FAD semiquinone simultaneously reduces ferredoxin. We have expressed the genes encodingThermotoga maritimaEtfABCX in E. coli and purified the EtfABCX holoenzyme and the EtfAB subcomplex. The bifurcation activity of EtfABCX was demonstrated by using electron paramagnetic resonance (EPR) to follow accumulation of reduced ferredoxin. To elucidate structural factors that impart the bifurcating ability, EPR and NADH titrations monitored by visible spectroscopy and dye-linked enzyme assays have been employed to characterize four conserved residues, R38, P239, and V242 in EtfA and R140 in EtfB, in the immediate vicinity of the BF-FAD. The R38, P239, and V242 variants showed diminished but still significant bifurcation activity. Despite still being partially reduced by NADH, the R140 variant had no bifurcation activity, and electron transfer to its two [4Fe-4S] clusters was prevented. The role of R140 is discussed in terms of the bifurcation mechanism in EtfABCX and in the other three families of bifurcating enzymes.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed. [ABSTRACT FROM AUTHOR]

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma -PK). Tma -PK was expressed in Escherichia coli and purified from the cells. Tma -PK exhibited higher thermostability than human PK. The purified Tma -PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma -PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma -PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus , the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents. [ABSTRACT FROM AUTHOR]

In bacteria, d -amino acids are primarily synthesized from l -amino acids by amino acid racemases, but some bacteria use d -amino acid aminotransferases to synthesize d -amino acids. d -Amino acids are peptidoglycan components in the cell wall involved in several physiological processes, such as bacterial growth, biofilm dispersal, and peptidoglycan metabolism. Therefore, their metabolism and physiological roles have attracted increasing attention. Recently, we identified novel bacterial d -amino acid metabolic pathways, which involve amino acid racemases, with broad substrate specificity, as well as multifunctional enzymes with d -amino acid-metabolizing activity. Here, I review these multifunctional enzymes and their related d - and l -amino acid metabolic pathways in Escherichia coli and the hyperthermophile Thermotoga maritima. [ABSTRACT FROM AUTHOR]

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. Key points: • β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima β-glucosidase has high activity and high thermostability. • Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

S-Adenosyl-l-homocysteine hydrolase (SAHH) reversibly cleaves S-adenosyl-l-homocysteine, the product of S-adenosyl-l-methionine-dependent methylation reactions. The conversion of S-adenosyl-l-homocysteine into adenosine and l-homocysteine plays an important role in the regulation of the methyl cycle. An alternative metabolic route for S-adenosyl-l-methionine regeneration in the extremophiles Methanocaldococcus jannaschii and Thermotoga maritima has been identified, featuring the deamination of S-adenosyl-l-homocysteine to S-inosyl-l-homocysteine. Herein, we report the structural characterisation of different archaeal SAHHs together with a biochemical analysis of various SAHHs from all three domains of life. Homologues deriving from the Euryarchaeota phylum show a higher conversion rate with S-inosyl-l-homocysteine compared to S-adenosyl-l-homocysteine. Crystal structures of SAHH originating from Pyrococcus furiosus in complex with SlH and inosine as ligands, show architectural flexibility in the active site and offer deeper insights into the binding mode of hypoxanthine-containing substrates. Altogether, the findings of our study support the understanding of an alternative metabolic route for S-adenosyl-l-methionine and offer insights into the evolutionary progression and diversification of SAHHs involved in methyl and purine salvage pathways. The biochemical characterisation of S-adenosyl-l-homocysteine hydrolases from all three domains of life supports the assumption that alternative routes for methyl metabolism coupled to purine salvage exist in some classes of Euryarchaeota. [ABSTRACT FROM AUTHOR]

Dynamic optimization of thermophilic hydrogen and by-product production requires the use of robust models coupled with control strategies. In this work, a model was used to optimize biohydrogen (bioH2) and acetate production using Thermotoga maritima (T. maritima) with a Multi-Objective Optimization (MOO). The aim was to find the trade-off between the maxima of yield and productivity of the hydrogen production and acetate in a continuous dark fermentation process by modifying the inlet liquid flow rate. A dynamic mass balance model was used to optimize thermophilic bioH2 and acetate production using T. maritima MSB8 (DSMZ 3109) strains. In this study, the simulations were run using an Intel® Core™ i7-9750 H @ 2.60 GHz, 16 GB RAM computer. The paretosearch integrated function from MATLAB® was used to obtain Pareto Optimal Sets (POSs) for each inlet substrate concentration. Some constraints were used to fit to the model dynamics of dark fermentation performed by T. maritima. using glucose as a substrate; the maximum theoretical hydrogen yield was 4 at 80 °C. The MOO was performed for five different substrate concentrations. The maximum Euclidean distance from the origin of the normalized coordinates was used to select the Pareto Optimal Points (POPs). The simulation results indicate that an equal to 70 × 10-3 is optimum to significantly maximize/minimize at 3.48 and 0.33 and at 3.49 × 10-3 and 1.66 × 10-3. h simultaneously, respectively, for hydrogen and acetate production. [ABSTRACT FROM AUTHOR]

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas. [ABSTRACT FROM AUTHOR]

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.

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.

Starch-like polymers can be created through the use of enzymatic modification with glycogen branching enzymes (GBEs). GBEs are categorized in the glycoside hydrolase (GH) family 13 and 57. Both GH13 and GH57 GBEs exhibit branching and hydrolytic activity. While GH13 GBEs are also capable of α-1,4-transglycosylation, it is yet unknown whether GH57 share this capability. Among the four crystal structures of GH57 GBEs that have been solved, a flexible loop with a conserved tyrosine was identified to play a role in the branching activity. However, it remains unclear whether this flexible loop is also involved in α-1,4-transglycosylation activity. We hypothesize that GH57 GBEs with the flexible loop and tyrosine are also capable of α-1,4-transglycosylation, similar to GH13 GBEs. The aim of the present study was to characterize the activity of GH57 GBEs to investigate a possible α-1,4-transglycosylation activity. Three GH57 GBEs were selected, one from Thermococcus kodakarensis with the flexible loop and two beta-strands; one from Thermotoga maritima, missing the flexible loop and beta-strands; and one from Meiothermus sp., missing the flexible loop but with the two beta-strands. The analysis of chain length distribution over time of modified maltooctadecaose, revealed, for the first time, that all three GH57 GBEs can generate chains longer than the substrate itself, showing that α-1,4-transglycosylation activity is generally present in GH57 GBEs. [ABSTRACT FROM AUTHOR]

In recent years, the synthesis of human milk oligosaccharides, particularly fucooligosaccharides, has been investigated. These oligosaccharides provide protection against gastrointestinal diseases for newborns, among other functionalities. α-L-fucosidase from Thermotoga maritima of GH29 family has been reported to produce fucooligosaccharides by means of a transfucosylation reaction. However, the interaction between acceptor and donor substrates, and the enzyme has been little studied. For that reason, and due to the importance of fucooligosaccharides, the present study proposes a theoretical synthesis route employing α-L-fucosidase from T. maritima, lactose as acceptor substrate and pNP-fucose as fucosyl donor. A molecular docking study was carried out using crystallographic structure of α-L-fucosidase from Protein Data Bank. By simulating the theoretical synthesis with T. maritima fucosidase, it was observed that lactose interacts with the enzyme in regions other than the active site. However, it is approaching the active site when the fucose is in the position to form the enzyme-substrate complex. This finding may explain the transfucosylation yield documented in the literature when the proportion of fucosyl acceptor substrate increases concerning that of the donor substrate. [ABSTRACT FROM AUTHOR]

Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria. [ABSTRACT FROM AUTHOR]

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP‐dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N‐terminal helicase domain of reverse gyrase with its C‐terminal type IA topoisomerase domain. This cooperation is mediated by a reverse‐gyrase‐specific insertion into the helicase domain termed the 'latch'. The latch consists of a globular domain inserted at the top of a β‐bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the β‐bulge loop is required for supercoiling activity. It has previously been shown that the β‐bulge loop constitutes a minimal latch that couples ATP‐dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β‐bulge loop as a minimal latch is reported. It is shown that the β‐bulge loop supports ATP‐dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk. [ABSTRACT FROM AUTHOR]

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 20-deoxynucleoside 50-monophosphates (dNMPs) and uridine 50-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T. maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (20-deoxy)nucleoside 50-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 50-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T. maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides. [ABSTRACT FROM AUTHOR]

Hydrogen (H2) is a promising energy carrier owing to its environmentally friendly properties and high energy content. The aim of this work is to achieve a continuous H2 production from a simplified culture medium composed of date wastes (DW) and natural seawater using the marine bacterium, Thermotoga maritima. Increasing the dilution rate (D) from 0.02 to 0.25 h− 1 in the continuous stirred tank reactor (CSTR) improved the volumetric H2 production rate (QH2) from 2 to 17 mmol/L.h. At a D above 0.25 h− 1, a washout of the bacterium cells was observed, resulting in a significant decrease of biomass concentration (BC) and then the drop in QH2. The production of H2 was improved by using a membrane bioreactor (MBR) due to the cell retention in the reactor. At a high D of 0.6 h− 1, the maximum QH2 was increased from 0.6 to 61.75 mmol/L.h, which is higher than those previously reported on continuous H2 production from different substrates. Moreover, increasing the hexose equivalent concentration in the date juice (DJ) from 30 to 60 mmol/L further enhanced the H2 production. It allowed a QH2 of 70.2 mmol/L.h which is interesting for a large scale production of H2 from date wastes. [ABSTRACT FROM AUTHOR]

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multiparameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multimutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.

The H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S]H-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe]H-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe]H-subcluster is assembled by a set of Fe-S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [FeII(Cys)(CO)2(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5'-deoxyadenosyl radical attacking the cysteine S to form a C5'-S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3-S bond, producing a mononuclear [FeI(CO)2(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe]H-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompa and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions. [ABSTRACT FROM AUTHOR]

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 U μmol−1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90 °C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60 °C for 1 h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling. [ABSTRACT FROM AUTHOR]

Oxidative alkene cleavage is a highly interesting reaction to obtain aldehydes and ketones. The Mn‐dependent protein TM1459 from Thermotoga maritima can catalyse alkene cleavage of styrene derivatives in the presence of tert‐butyl hydroperoxide. Despite the high thermal stability of the enzyme, it gets inactivated during the reaction. The data reported here indicate that auto‐oxidation is responsible for the low stability of TM1459 in the oxidative environment required for the alkene cleavage reaction. By targeting the exchange of residues prone to oxidation, this phenomenon was successfully prevented. Importantly, the stability to oxidation conveyed by the amino acid exchanges led to increased enzyme activity. However, the exchanges resulted in slightly modified positions of two of the four metal‐binding amino acids, thereby strongly impacting metal binding. [ABSTRACT FROM AUTHOR]

Thermotoga maritima is a hypertherinophilic, anaerobic organism. As a member of the order Thermo - togates, T maritima carry a unique ability as only fermentative bacterium to produce hydrogen as clean energy. We noticed a hypothetical protein TM] 070 [Protein Data Bank, PDB 1NC71. This protein is characterized as a member of DUE domain of unknown function. Interestingly. this sequence length of 138 amino acids matches to only known structure of cognate transducer in Anabaena PCC 7120 [2Ii'7-21I9 etc]. The scaffold of both transducer and TM 1070 protein is tetrameric. We analyzed 114 highly conserved sequence two these two proteins. The detailed structural analysis of all three space groups (P4, (2 and P21. 21,21-large crystal) transducer forms the same tightly packed ten·ainer with C4 symmetry. In the P4 space group, this tetramer is formed by crystallographic symmetry, while in the other two space groups the tetramer syinmetry is non-crystallographic. The same tetramer was observed in the only structural homolog of transducer iii the PDB, protein structure of 1 NC7. This 1 14 residue protein possesses significant sequence identity with transducer. In this project we have focused on motif structural insight using PyMol, AxPyMol and NovaFold A I. Interestingly, we found well-coordinated divalent magnesium present in the core of tetrameric assesmbly of INC7 with polar pocket [Thr amino acid]. It is likely that magnesium is crucial for phosphory] transfer, as found in transducer leading to destabilization of tetramer as part of signaling state. Our detailed motif characterization reveal a novel ligand binding to this DUF tetramer. [ABSTRACT FROM AUTHOR]

Scaffold-based protein libraries are designed to be both diverse and rich in functional/folded proteins. However, introducing an extended diversity while preserving stability of the initial scaffold remains a challenge. Here we developed an original approach to select the ensemble of folded proteins from an initial library. The thermostable CheY protein from Thermotoga maritima was chosen as scaffold. Four loops of CheY were diversified to create a new binding surface. The subset of the library giving rise to folded proteins was first selected using a natural protein partner of the template scaffold. Then, a gene shuffling approach based on a single restriction enzyme was used to recombine DNA sequences encoding these filtrated variants. Taken together, the filtration strategy and the shuffling of the filtrated sequences were shown to enrich the library in folded and stable sequences while maintaining a large diversity in the final library (Lib-Cheytins 2.1). Binders of the Oplophorus luciferase Kaz domain were then selected by phage display from the final library, showing affinities in the μM range. One of the best variants induced a loss of 92% of luminescent activity, suggesting that this Cheytin preferentially binds to the Kaz active site. [ABSTRACT FROM AUTHOR]

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size. Here, the crystal structures of two permuted halves of a modern ribose‐binding protein (RBP) from Thermotoga maritima are reported. The overexpressed proteins are well folded and show a monomer–dimer equilibrium in solution. Their crystal structures show partially noncanonical PBP‐like fold type I conformations with structural deviations from modern RBPs. One of the half variants forms a dimer via segment swapping, suggesting a high degree of malleability. The structural findings on these permuted halves support the evolutionary hypothesis that PBPs arose via a duplication event of a flavodoxin‐like protein and further support a domain‐swapping step that might have occurred during the evolution of the PBP‐like fold, a process that is necessary to generate the characteristic motion of PBPs essential to perform their functions. [ABSTRACT FROM AUTHOR]

The basis of MreB research is the study of the MreB protein from the Thermotoga maritima species, since it was the first one whose crystal structure was described. Since MreB proteins from different bacterial species show different polymerisation properties in terms of nucleotide and salt dependence, we conducted our research in this direction. For this, we performed measurements based on tryptophan emission, which were supplemented with temperature-dependent and chemical denaturation experiments. The role of nucleotide binding was studied through the fluorescent analogue TNP-ATP. These experiments show that Thermotoga maritima MreB is stabilised in the presence of low salt buffer and ATP. In the course of our work, we developed a new expression and purification procedure that allows us to obtain a large amount of pure, functional protein. [ABSTRACT FROM AUTHOR]

The first hyperthermophilic L-arabinose/D-galactose 1-dehydrogenase (TmAraDH) from Thermotoga maritima was heterologously purified from Escherichia coli. It belongs to the Gfo/Idh/MocA protein family, prefers NAD+/NADP+ as a cofactor. The purified TmAraDH exhibited maximum activity toward L-arabinose at 75 °C and pH 8.0, and retained 63.7% of its activity after 24 h at 60 °C, and over 60% of its activity after holding a pH ranging from 7.0 to 9.0 for 1 h. Among all tested substrates, TmAraDH exclusively catalyzed the NAD(P)+-dependent oxidation of L-arabinose, D-galactose and D-fucose. The catalytic efficiency (kcat/Km) towards L-arabinose and D-galactose was 123.85, 179.26 min−1 mM−1 for NAD+, and 56.06, 18.19 min−1 mM−1 for NADP+, respectively. TmAraDH exhibited complete oxidative conversion in 12 h at 70 °C to D-galactonate with 5 mM D-galactose. Modelling provides structural insights into the cofactor and substrate recognition specificity. Our results suggest that TmAraDH have great potential for the conversion of L-arabinose and D-galactose to L-arabonate and D-galactonate. [ABSTRACT FROM AUTHOR]

Biohydrogen production by the anaerobic hyperthermophilic and halophilic bacterium, Thermotoga maritima (TM), was conducted using a mixture of Onion Waste Juice (OWJ) and seawater (SW). The highest production of biohydrogen (H2) with OWJ, as the exclusive source of carbon and energy, was obtained for an optimum volume of 50% (v/v), with the highest overall productivity of biohydrogen (15.6 mM/h) and a maximum yield of 2.6 ($\mathrm{mol}_{\mathrm{H}_{2}}/\mathrm{mol}_{\mathrm{Hexose}}$). This was mainly due to the presence of organosulfur compounds and the natural presence of ammonium contained in OWJ. The addition of inorganic nitrogen and iron sources in the mixture of SW and OWJ has improved biohydrogen production, achieving productivity yield (23.0 mM/h for 3.2 $\mathrm{mol}_{\mathrm{H}_{2}}/\mathrm{mol}_{\mathrm{Hexose}}$) close to the maximum obtained for TM. Above 600 mL, the high concentration of substrate ($>$30 gCOD/L) led the metabolism to deviate towards lactate production at the expense of H2 production. A fed-batch culture with the sequential addition of concentrated OWJ mixed with only sea salt was investigated for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The total cumulative biohydrogen produced was about 300 mM after 30 h of incubation.

Numerous studies have shown how periplasmic binding proteins (PBPs) bind substrates with exquisite specificity, even distinguishing between sugar epimers and anomers, or structurally similar ions. Yet, marked substrate promiscuity is also a feature encoded in some PBPs. Except for three sub-Ångström crystal structures, there are no reports of hydrogen atom positions in the remaining (> 1000) PBP structures. The previous X-ray crystal structure of the maltodextrin periplasmic-binding protein from Thermotoga maritima (tmMBP) complexed with oligosaccharide showed a large network of interconnected water molecules stretching from one end of the substrate binding pocket to the other. These water molecules are positioned to form multiple hydrogen bonds, as well as forming interactions between the protein and substrate. Here we present the neutron crystal structure of tmMBP to a resolution of 2.1 Å. This is the first neutron crystal structure from the PBP superfamily and here we unambiguously identify the nature and orientation of the hydrogen bonding and water-mediated interactions involved in stabilizing a tetrasaccharide in the binding site. More broadly, these results demonstrate the conserved intricate mechanisms that underlie substrate-specificity and affinity in PBPs. [ABSTRACT FROM AUTHOR]

Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies. Author summary: In this work, we demonstrated conservation of lipid-interaction sites on proteins from two species that deviated from their evolutionary common ancestors a long time ago, using the membrane-integral pyrophosphatases from a thermophilic bacteria species and a plant species. We further showed that these sites are preserved in homology modelled proteins of the same family. This retention of a common lipid interaction profile or "fingerprint" and our ability to predict this for other proteins in this family may indicate that they are more integral to protein function than previously thought. By identifying lipid interactions that may act to stabilise the protein structure, these properties could be exploited to solve protein structures, and the interfacial site's potential involvement in inter-subunit communication may be useful for further investigation of the catalytic cycle of this clinically relevant membrane protein family. Word Count: 140 [ABSTRACT FROM AUTHOR]

d‐Amino acids (d‐AAs) are key components of the peptidoglycan matrix in bacterial cells. Various bacterial species are known to produce d‐AAs by using different enzymes, such as highly specific and broad‐spectrum racemases. Miyamoto et al. studied the biosynthesis of d‐glutamate in the hyperthermophile and anaerobic Gram‐negative bacterium, Thermotoga maritima, which does not possess a broad‐spectrum racemase. The investigated TM0831 enzyme catalyzes both a d‐amino acid aminotransferase reaction producing d‐glutamate and an amino acid racemase activity aimed at generating d‐aspartate and d‐glutamate from the corresponding l‐enantiomers. TM0831 represents an example of natural molecular evolution process favoring the enzyme versatility. Comment on: https://doi.org/10.1111/febs.16452 [ABSTRACT FROM AUTHOR]

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d‐lysine alongside the usual d‐alanine and d‐glutamate. We previously identified a lysine racemase involved in d‐lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d‐alanine and d‐glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d‐amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d‐amino acids, but did not display activity towards l‐amino acids. It displayed high specific activities towards d‐homoserine and d‐glutamine as amino donors. The most preferred acceptor was 2‐oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat/Km) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)‐deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831‐integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild‐type strain. These results suggest that TM0831 is involved in d‐glutamate production. TM0831 is a novel d‐amino acid aminotransferase with racemase activity that is involved in the production of d‐amino acids in T. maritima. [ABSTRACT FROM AUTHOR]

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF. [ABSTRACT FROM AUTHOR]

Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site. [ABSTRACT FROM AUTHOR]

Biohydrogen production by the anaerobic hyperthermophilic and halophilic bacterium, Thermotoga maritima (TM), was conducted using a mixture of OnionWaste Juice (OWJ) and seawater (SW). The highest production of biohydrogen (H2) with OWJ, as the exclusive source of carbon and energy, was obtained for an optimum volume of 50% (v/v), with the highest overall productivity of biohydrogen (15.6 mM/h) and a maximum yield of 2.6 (molH2/molHexose). This was mainly due to the presence of organosulfur compounds and the natural presence of ammonium contained in OWJ. The addition of inorganic nitrogen and iron sources in the mixture of SW and OWJ has improved biohydrogen production, achieving productivity yield (23.0 mM/h for 3.2 molH2/molHexose) close to the maximum obtained for TM. Above 600 mL, the high concentration of substrate (>30 gCOD/L) led the metabolism to deviate towards lactate production at the expense of H2 production. A fed-batch culture with the sequential addition of concentrated OWJ mixed with only sea salt was investigated for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The total cumulative biohydrogen produced was about 300mMafter 30 h of incubation. [ABSTRACT FROM AUTHOR]

Thermophiles and hyperthermophiles are immensely useful in understanding the evolution of life, besides their utility in environmental and industrial biotechnology. Advancements in sequencing technologies have revolutionized the field of microbial genomics. The massive generation of data enhances the sequencing coverage multi-fold and allows to analyse the entire genomic features of microbes efficiently and accurately. The mandate of a pure isolate can also be bypassed where whole metagenome-assembled genomes and single cell-based sequencing have fulfilled the majority of the criteria to decode various attributes of microbial genomes. A boom has, therefore, been seen in analysing the extremophilic bacteria and archaea using sequence-based approaches. Due to extensive sequence analysis, it becomes easier to understand the gene flow and their evolution among the members of bacteria and archaea. For instance, sequencing unveiled that Thermotoga maritima shares around 24% of genes of archaeal origin. Comparative and functional genomics provide an analytical view to understanding the microbial diversity of thermophilic bacteria and archaea, their interactions with other microbes, their adaptations, gene flow, and evolution over time. In this review, the genomic features of thermophilic bacteria and archaea are dealt with comprehensively. [ABSTRACT FROM AUTHOR]

In the current study, the purified β-mannanase (Man/Cel5B) from Thermotoga maritima was immobilized on glutaraldehyde cross-linked chitosan beads. The immobilization of Man/Cel5B on chitosan beads was confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. After immobilization, the protein loading efficiency and immobilization yield were found to be 73.3% and 71.8%, respectively. The optimum pH for both free and immobilized enzymes was found to be pH 5.5. However, the optimum temperature of immobilized Man/Cel5B increased by 10 °C, from 85 °C (free Man/Cel5B) to 95 °C (Immobilized). The half-life of free and immobilized enzymes was found to be 7 h and 9 h, respectively, at 85 °C owing to the higher thermostability of immobilized Man/Cel5B. The increase in thermostability was also demonstrated by an increase in the energy of deactivation (209 kJmol−1) for immobilized enzyme compared to its native form (92 kJmol−1), at 85 °C. Furthermore, the immobilized Man/Cel5B displayed good operational stability as it retained 54% of its original activity after 15 repeated catalytic reactions concerning its free form. [ABSTRACT FROM AUTHOR]

Sulfur-insertion reactions are essential for the biosynthesis of several cellular metabolites, including enzyme cofactors. In Lactobacillus plantarum, a sulfur-containing nickel-pincer nucleotide (NPN) cofactor is used as a coenzyme of lactic acid racemase, LarA. During NPN biosynthesis in L. plantarum, sulfur is transferred to a nicotinic acid-derived substrate by LarE, which sacrifices the sulfur atom of its single cysteinyl side chain, forming a dehydroalanine residue. Most LarE homologs contain three conserved cysteine residues that are predicted to cluster at the active site; however, the function of this cysteine cluster is unclear. In this study, we characterized LarE from Thermotoga maritima (LarETm) and show that it uses these three conserved cysteine residues to bind a [4Fe-4S] cluster that is required for sulfur transfer. Notably, we found LarETm retains all side chain sulfur atoms, in contrast to LarELp. We also demonstrate that when provided with L-cysteine and cysteine desulfurase from Escherichia coli (IscSEc), LarETm functions catalytically with IscSEc transferring sulfane sulfur atoms to LarETm. Native mass spectrometry results are consistent with a model wherein the enzyme coordinates sulfide at the nonligated iron atom of the [4Fe-4S] cluster, forming a [4Fe-5S] species, and transferring the noncore sulfide to the activated substrate. This proposed mechanism is like that of TtuA that catalyzes sulfur transfer during 2-thiouridine synthesis. In conclusion, we found that LarE sulfur insertases associated with NPN biosynthesis function either by sacrificial sulfur transfer from the protein or by transfer of a noncore sulfide bound to a [4Fe-4S] cluster. [ABSTRACT FROM AUTHOR]

The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N terminal domain of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliFC induces substantial conformational changes in FliGN and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliFC:FliGN complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane.

BackgroundThe success of genome-scale models (GEMs) can be attributed to the high-quality, bottom-up reconstructions of metabolic, protein synthesis, and transcriptional regulatory networks on an organism-specific basis. Such reconstructions are biochemically, genetically, and genomically structured knowledge bases that can be converted into a mathematical format to enable a myriad of computational biological studies. In recent years, genome-scale reconstructions have been extended to include protein structural information, which has opened up new vistas in systems biology research and empowered applications in structural systems biology and systems pharmacology.ResultsHere, we present the generation, application, and dissemination of genome-scale models with protein structures (GEM-PRO) for Escherichia coli and Thermotoga maritima. We show the utility of integrating molecular scale analyses with systems biology approaches by discussing several comparative analyses on the temperature dependence of growth, the distribution of protein fold families, substrate specificity, and characteristic features of whole cell proteomes. Finally, to aid in the grand challenge of big data to knowledge, we provide several explicit tutorials of how protein-related information can be linked to genome-scale models in a public GitHub repository ( https://github.com/SBRG/GEMPro/tree/master/GEMPro_recon/).ConclusionsTranslating genome-scale, protein-related information to structured data in the format of a GEM provides a direct mapping of gene to gene-product to protein structure to biochemical reaction to network states to phenotypic function. Integration of molecular-level details of individual proteins, such as their physical, chemical, and structural properties, further expands the description of biochemical network-level properties, and can ultimately influence how to model and predict whole cell phenotypes as well as perform comparative systems biology approaches to study differences between organisms. GEM-PRO offers insight into the physical embodiment of an organism's genotype, and its use in this comparative framework enables exploration of adaptive strategies for these organisms, opening the door to many new lines of research. With these provided tools, tutorials, and background, the reader will be in a position to run GEM-PRO for their own purposes.

Most known cellulase‐associated carbohydrate‐binding modules (CBMs) are attached to the N‐ or C‐terminus of the enzyme or are expressed separately and assembled into multi‐enzyme complexes (for example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR‐220, a GH5‐family endo‐β‐1,4‐glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A‐like catalytic domain with a (β/α)8 TIM‐barrel fold and an inserted CBM29‐like noncatalytic domain with a β‐jelly‐roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full‐length and CBM‐deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full‐length and CBM‐deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single‐chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross‐domain groove. Subsequent site‐specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase. [ABSTRACT FROM AUTHOR]

The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (Kd). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the Kd at four different temperatures. These Kd values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the Kd determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the Kd caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the Kd, can be carried out using common laboratory equipment and is scalable. [ABSTRACT FROM AUTHOR]

The inter-subunit interaction at the protein interfaces plays a key role in protein self-assembly, through which enabling protein self-assembly controllable is of great importance for preparing the novel nanoscale protein materials with unexplored properties. Different from normal 24-meric ferritin, archaeal ferritin, Thermotoga maritima ferritin (TmFtn) naturally occurs as a dimer, which can assemble into a 24-mer nanocage induced by salts. However, the regulation mechanism of protein self-assembly underlying this phenomenon remains unclear. Here, a combination of the computational energy simulation and key interface reconstruction revealed that a short helix involved interactions at the C 4 interface are mainly responsible for the existence of such dimer. Agreeing with this idea, deletion of such short helix of each subunit triggers it to be a stable dimer, which losses the ability to reassemble into 24-meric ferritin in the presence of salts in solution. Further support for this idea comes from the observation that grafting a small helix from human H ferritin onto archaeal subunit resulted in a stable 24-mer protein nanocage even in the absence of salts. Thus, these findings demonstrate that adjusting the interactions at the protein interfaces appears to be a facile, effective approach to control subunit assembly into different protein architectures. [Display omitted] • Computational analyses reveal the energy difference between HuHF and TmFtn. • A short helix is crucial for TmFtn assembly from dimer to 24-mer. • Deletion of the short helix makes TmFtn only stay in its dimeric form. • Grafting E -helix of HuHF onto TmFtn enables it to only stay in its 24meric form. [ABSTRACT FROM AUTHOR]

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx. [ABSTRACT FROM AUTHOR]

Introduction: An attractive and novel proposal for the application of enzymatic inversion of sucrose on industrial scale is constituted by ß-fructosidase from Thermotoga maritima (BfrA) expressed in Pichia pastoris and immobilized on calcium alginate (PpABfrA), because the high thermostability and thermoactivity of this biocatalyst. Objective: To determine the operational conditions for the industrial scale-up of enzymatic hydrolysis of sucrose in a glucose production facility, based on the optimal experimental variables and the requirements for auxiliary facilities. Materials and Methods: Design of experiments and optimization of the behavior of the experimental variables influencing the percentage of hydrolysis achieved with the thermostable biocatalyst PpABfrA were combined. The experimental reactive conditions were scaled and the technology for this inversion method insertion was selected, with its corresponding consumptions. Results and Discussion: The optimum reaction time was 12 h for a sucrose concentration of 1.75 M, where 85.6 % inversion is achieved. In turn, keeping the biocatalyst shear constant in the reactor was selected as the most successful scaling criterion. With the technological conception of this stage, a hydrolysis capacity of 201.6 t of sucrose in 11 days was estimated, where 16 inversion cycles are carried out with 2.10 t of biocatalyst, in an effective reaction volume of 24 m³. Conclusions: The operational parameters determined on industrial scale for the insertion of this technology, ensure an increase the yield and quality of obtained glucose. [ABSTRACT FROM AUTHOR]

AAA+ proteases regulate numerous physiological and cellular processes through tightly regulated proteolytic cleavage of protein substrates driven by ATP hydrolysis. FtsH is the only known family of membrane-anchored AAA+ proteases essential for membrane protein quality control. Although a spiral staircase rotation mechanism for substrate translocation across the FtsH pore has been proposed, the detailed conformational changes among various states have not been clear due to absence of FtsH structures in these states. We report here the cryo-EM structure for Thermotoga maritima FtsH (TmFtsH) in a fully ADP-bound symmetric state. Comparisons of the ADP-state structure with its apo-state and a substrate-engaged yeast YME1 structure show conformational changes in the ATPase domains, rather than the protease domains. A reconstruction of the full-length TmFtsH provides structural insights for the dynamic transmembrane and the periplasmic domains. Our structural analyses expand the understanding of conformational switches between different nucleotide states in ATP hydrolysis by FtsH. Liu et. al determine the cryo-EM structure of ADP-bound transmembrane AAA+ protease FtsH and provide insights into conformational switches between different nucleotide states important for ATP hydrolysis by FtsH. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)