Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis.

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N    5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na+ transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B12 derivative (cobamide) as prosthetic group. We present the 2.08 Šcryo-EM structure of Mtr(ABCDEFG)3 composed of the central Mtr(ABFG)3 stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na+ were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na+ transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B12, the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na+ flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na+ outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B12-carrying domain.

Growth of carbon nanotubes over carbon nanofibers catalyzed by bimetallic alloy nanoparticles as a bifunctional electrode for Zn-air batteries.

Design of economical, large-scale, stable, and highly active bifunctional electrocatalysts for Zn-air batteries with enhanced oxygen reduction and oxygen evolution performance is needed. Herein, a series of electrocatalysts were facilely fabricated where in situ formed bimetallic nanoparticles aided in the growth of carbon nanotubes over carbon nanofibers (MM'-CNT@CNF) during thermal treatment. Different combinations of Fe, Ni, Co and Mn metals and melamine as precursor for CNT growth were investigated. The synergistic interaction between bimetallic nanoparticles and N-doped carbon results in greatly improved bifunctional catalytic activity for both oxygen reduction and evolution reactions (ORR, OER) using FeNi-CNT@CNF as catalyst. The half-wave potential (0.80 V vs. RHE) for FeNi-CNT@CNF for ORR was close to that of Pt/C (0.79 V vs. RHE), meanwhile its stability was superior to Pt/C. Likewise, during OER, the FeNi-CNT@CNF reached a current density of 10 mA cm-2 at a rather low overpotential of 310 mV vs. RHE compared to benchmark RuO2 (410 mV). The rechargeable Zn-air prototype battery using FeNi-CNT@CNF as an air electrode outperformed the mixture of Pt/C and RuO2 with discharge/charge overpotential of 0.61 V, power density of 118 mW cm-2 at 10 mA cm-2 and an improved cycling stability over 108 hours.

The molybdenum storage protein forms and deposits distinct polynuclear tungsten oxygen aggregates.

Some N2-fixing bacteria store Mo to maintain the formation of the vital FeMo-cofactor dependent nitrogenase under Mo depleting conditions. The Mo storage protein (MoSto), developed for this purpose, has the unique capability to compactly deposit molybdate as polyoxometalate (POM) clusters in a (αß)3 hexameric cage; the same occurs with the physicochemically related tungstate. To explore the structural diversity of W-based POM clusters, MoSto loaded under different conditions with tungstate and two site-specifically modified MoSto variants were structurally characterized by X-ray crystallography or single-particle cryo-EM. The MoSto cage contains five major locations for POM clusters occupied among others by heptanuclear, Keggin ion and even Dawson-like species also found in bulk solvent under defined conditions. We found both lacunary derivatives of these archetypical POM clusters with missing WOx units at positions exposed to bulk solvent and expanded derivatives with additional WOx units next to protecting polypeptide segments or other POM clusters. The cryo-EM map, unexpectedly, reveals a POM cluster in the cage center anchored to the wall by a WOx linker. Interestingly, distinct POM cluster structures can originate from identical, highly occupied core fragments of three to seven WOx units that partly correspond to those found in MoSto loaded with molybdate. These core fragments are firmly bound to the complementary protein template in contrast to the more variable, less occupied residual parts of the visible POM clusters. Due to their higher stability, W-based POM clusters are, on average, larger and more diverse than their Mo-based counterparts.

Electrospun aluminum silicate nanofibers as novel support material for immobilization of alcohol dehydrogenase.

Ceramic materials with high surface area, large and open porosity are considered excellent supports for enzyme immobilization owing to their stability and reusability. The present study reports the electrospinning of aluminum silicate nanofiber supports from sol-gel precursors, the impact of different fabrication parameters on the microstructure of the nanofibers and their performance in enzyme immobilization. A change in nanofiber diameter and pore size of the aluminum silicate nanofibers was observed upon varying specific processing parameters, such as the sol-composition (precursor and polymer concentration), the electrospinning parameters and the subsequent heat treatment (calcination temperature). The enzyme, alcohol dehydrogenase (ADH), was immobilized on the aluminum silicate nanofibers by physical adsorption and covalent bonding. Activity retention of 17% and 42% was obtained after 12 d of storage and repeated reaction cycles for physically adsorbed and covalently bonded ADH, respectively. Overall, the immobilization of ADH on aluminum silicate nanofibers resulted in high enzyme loading and activity retention. However, as compared to covalent immobilization, a marked decrease in the enzyme activity during storage for physically adsorbed enzymes was observed, which was ascribed to leakage of the enzymes from the nanofibers. Such fibers can improve enzyme stability and promote a higher residual activity of the immobilized enzyme as compared to the free enzyme. The results shown in this study thus suggest that aluminum silicate nanofibers, with their high surface area, are promising support materials for the immobilization of enzymes.

Nanostructured carbons containing FeNi/NiFe2O4 supported over N-doped carbon nanofibers for oxygen reduction and evolution reactions.

Non-precious metal-based electrocatalysts on carbon materials with high durability and low cost have been developed to ameliorate the oxygen-reduction reaction (ORR) and oxygen-evolution reaction (OER) for electrochemical energy applications such as in fuel cells and water electrolysis. Herein, two different morphologies of FeNi/NiFe2O4 supported over hierarchical N-doped carbons were achieved via carbonization of the polymer nanofibers by controlling the ratio of metal salts to melamine: a mixture of carbon nanotubes (CNTs) and graphene nanotubes (GNTs) supported over carbon nanofibers (CNFs) with spherical FeNi encapsulated at the tips (G/CNT@NCNF, 1 : 3), and graphene sheets wrapped CNFs with embedded needle-like FeNi (GS@NCNF, 2 : 3). G/CNT@NCNF shows excellent ORR activity (on-set potential: 0.948 V vs. RHE) and methanol tolerance, whilst GS@NCNF exhibited significantly lower over-potential of only 230 mV at 10 mA cm-2 for OER. Such high activities are due to the synergistic effects of bimetallic NPs encapsulated at CNT tips and N-doped carbons with unique hierarchical structures and the desired defects.

A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km ) toward R1P was 11.5 mM-1 · s-1 ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis.

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

Glutathione-Dependent Formaldehyde Dehydrogenase Homolog from Bacillus subtilis Strain R5 is a Propanol-Preferring Alcohol Dehydrogenase.

Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathione-dependent formaldehyde dehydrogenase.

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

Pcal_1127, a highly stable and efficient ribose-5-phosphate pyrophosphokinase from Pyrobaculum calidifontis.

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.