Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Antiterminator LoaP loads onto RNA to chase a runaway RNA polymerase.

In this issue of Structure, Elghondakly et al.1 present the crystal structure of Thermoanaerobacter pseudethanolicus antiterminator LoaP, a member of a ubiquitous family of NusG transcription factors, bound to its target, a dfn RNA hairpin. LoaP uses RNA as a recognition determinant, which is unique among NusG paralogs and makes unusual contacts in the major groove of the RNA.

Industrial biotechnology goes thermophilic: Thermoanaerobes as promising hosts in the circular carbon economy.

Transitioning away from fossil feedstocks is imperative to mitigate climate change, and necessitates the utilization of renewable, alternative carbon and energy sources to foster a circular carbon economy. In this context, lignocellulosic biomass and one-carbon compounds emerge as promising feedstocks that could be renewably upgraded by thermophilic anaerobes (thermoanaerobes) via gas fermentation or consolidated bioprocessing to value-added products. In this review, the potential of thermoanaerobes for cost-efficient, effective and sustainable bioproduction is discussed. Metabolic and bioprocess engineering approaches are reviewed to draw a comprehensive picture of current developments and future perspectives for the conversion of renewable feedstocks to chemicals and fuels of interest. Selected bioprocessing scenarios are outlined, offering practical insights into the applicability of thermoanaerobes at a large scale. Collectively, the potential advantages of thermoanaerobes regarding process economics could facilitate an easier transition towards sustainable bioprocesses with renewable feedstocks.

Major-groove sequence-specific RNA recognition by LoaP, a paralog of transcription elongation factor NusG.

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding ß-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family.

Designing Michaelases: exploration of novel protein scaffolds for iminium biocatalysis.

Biocatalysis is becoming a powerful and sustainable alternative for asymmetric catalysis. However, enzymes are often restricted to metabolic and less complex reactivities. This can be addressed by protein engineering, such as incorporating new-to-nature functional groups into proteins through the so-called expansion of the genetic code to produce artificial enzymes. Selecting a suitable protein scaffold is a challenging task that plays a key role in designing artificial enzymes. In this work, we explored different protein scaffolds for an abiological model of iminium-ion catalysis, Michael addition of nitromethane into E-cinnamaldehyde. We studied scaffolds looking for open hydrophobic pockets and enzymes with described binding sites for the targeted substrate. The proteins were expressed and variants harboring functional amine groups - lysine, p-aminophenylalanine, or N6-(D-prolyl)-L-lysine - were analyzed for the model reaction. Among the newly identified scaffolds, a thermophilic ene-reductase from Thermoanaerobacter pseudethanolicus was shown to be the most promising biomolecular scaffold for this reaction.

Revealing the role of the X25 domains through the characterization of truncated variants of amylopullulanase enzyme from Thermoanaerobacter brockii brockii.

To understand the role of the X25 domains of the amylopullulanase enzyme from Thermoanaerobacter brockii brockii (T. brockii brockii), four truncated variants that are TbbApuΔX25-1-SH3 (S130-A1484), TbbApuΔX25-2-SH3 (T235-A1484), TbbApuΔX25-1-CBM20 (S130-P1254), and TbbApuΔX25-2-CBM20 (T235-P1254) were constructed, expressed and characterized together with the SH3 and CBM20 domain truncated variants (TbbApuΔSH3 (V1-A1484) and TbbApuΔCBM20 (V1-P1254). TbbApuΔSH3 showed improved affinity and specificity for both pullulan and soluble starch than full-length TbbApu with lower Km and higher kcat/Km values. It indicates that SH3 is a disposable domain without any effect on the activity and stability of the enzyme. However, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20, TbbApuΔX25-2-CBM20 (T235-P1254) and TbbApuΔCBM20 showed higher Km and lower kcat/Km values than TbbApuΔSH3 to both soluble starch and pullulan. It specifies that the X25 domains and CBM20 play an important role in both α-amylase and pullulanase activity. Also, it is revealed that while truncation of the CBM20 domain as starch binding domain (SBD) did not affect on raw starch binding ability of the enzyme, truncation of both X25 domains caused almost complete loss of the raw starch binding ability of the enzyme. All these results enlightened the function of the X25 domains that play a more crucial role than CBM20 in the enzyme's binding to raw starch and also play a crucial role in its activity.

The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates.

Thermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66°C) and with high growth rates on hydrogen (H2) plus carbon dioxide (CO2). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H2 + CO2. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H2 may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech's is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H2 + CO2. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H2-dependent Fd reduction during growth on sugar substrates, putatively the H2-dependent CO2 reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While ∆ech2 grew well on pyruvate after four transfers, ∆ech2 did not adapt to CO. Cell suspensions of ∆ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate. IMPORTANCE: Acetogens thrive by converting H2+CO2 to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G'<-20 kJ mol-1). CO2 serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H2 + CO2 is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold-energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H2 to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications.

A Versatile Aldehyde: Ferredoxin Oxidoreductase from the Organic Acid Reducing Thermoanaerobacter sp. Strain X514.

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

Role of the C-Terminal ß Sandwich of Thermoanaerobacter tengcongensis Thermophilic Esterase in Hydrolysis of Long-Chain Acyl Substrates.

To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.

Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2.

Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Cloning and characterization of thermostable amylopullulanase TbbApu and its C-terminal truncated variants with enhanced activity in organic solvents.

Bifunctional debranching-enzyme amylopullulanases belong to the glycoside hydrolases (GHs) family and catalyze both the hydrolysis of α-1,4 and α-1,6 glycosidic bonds in starch, pullulan, amylopectin and glycogen polysaccharides. Among these, especially thermostable ones are essential in starch processing applications. In this study, we focused to elucidate the complete sequence of the apu gene and the role of C-term domains on biochemical properties and enzyme activity of Thermoanaerobacter brockii brockii amylopullulanase (TbbApu). After the gene sequence was defined, C- term truncated variants were constructed. The most suitable host organism and expression vector were determined as E. coli BL21(DE3) and pET-28a(+) depending on the highest yield/biomass ratio for recombinant production of all constructs. It was seen that the expression yield increased approximately threefold in the case of the SH3 region truncation. In the biochemical characterization, TbbApu and its truncated variants exhibited maximum activity at 70 °C and 75 °C for pullulan and starch hydrolysis respectively, and the optimum pH of TbbApu were 6.5 and 6 for truncated variants. Moreover, hydrolysis activities of all recombinant enzymes were enhanced by Mn2+, Co2+ and Cu2+, detergents, and almost all organic solvents; except butanol, DMF and DMSO. All recombinant amylopullulanases remained 80% stable up to 80 °C in the wide range of pH and also retained > 85% stability in the presence of defined volatile organic solvents. No significant difference was observed between the raw starch adsorption capacity and the specific activity of the three variants. These results indicated that the C-terminal regions of TbbApu are non-essential for the enzyme activity, stability and substrate binding capacity; furthermore, hexane and acetone organic solvents enhanced both pullulanase and α-amylase activity of these enzymes, interestingly. With these features, TbbApu and its truncated variants are distinguished from other thermophilic amylopullulanases and also make them promising candidates for industrial use.

Identification, molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus.

PURPOSE: We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family 15 of glycosylhydrolases (GH15). METHODS: The expression of this enzyme was optimized in E. coli (BL21) cells in order to have the highest amount of soluble protein (around 3 mg/l of culture medium). RESULTS: TeGA showed a high optimum temperature of 75 °C. It also showed one of the highest specific activities reported for a bacterial glucoamylase (75.3 U/mg) and was also stable in a wide pH range (3.0-10.0). Although the enzyme was preferentially active with maltose, it was also able to hydrolyze different soluble starches such as those from potato, corn or rice. TeGA showed a high thermostability up to around 70 °C, which was increased in the presence of PEG8000, and also showed to be stable in the presence of moderate concentrations of ethanol. CONCLUSION: We propose that TeGA could be suitable for use in different industrial processes such as biofuel production and food processing.

Membrane-anchored HDCR nanowires drive hydrogen-powered CO2 fixation.

Filamentous enzymes have been found in all domains of life, but the advantage of filamentation is often elusive1. Some anaerobic, autotrophic bacteria have an unusual filamentous enzyme for CO2 fixation-hydrogen-dependent CO2 reductase (HDCR)2,3-which directly converts H2 and CO2 into formic acid. HDCR reduces CO2 with a higher activity than any other known biological or chemical catalyst4,5, and it has therefore gained considerable interest in two areas of global relevance: hydrogen storage and combating climate change by capturing atmospheric CO2. However, the mechanistic basis of the high catalytic turnover rate of HDCR has remained unknown. Here we use cryo-electron microscopy to reveal the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui. The minimum repeating unit is a hexamer that consists of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of hydrogenase Fe-S subunits, one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize through their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we show that filamentation and rapid electron transfer through the filament enhance the activity of HDCR. To investigate the structure of HDCR in situ, we imaged T. kivui cells with cryo-electron tomography and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance the stability and connectivity of HDCR to form a specialized metabolic subcompartment within the cell.

A purified energy-converting hydrogenase from Thermoanaerobacter kivui demonstrates coupled H+-translocation and reduction in vitro.

Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N'-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.

Carbohydrate-binding module of cycloisomaltooligosaccharide glucanotransferase from Thermoanaerobacter thermocopriae improves its cyclodextran production.

Thermoanaerobacter thermocopriae-derived thermostable cycloisomaltooligosaccharide (CI)-forming enzymes catalyze the production of CIs from dextran. The primary structure of the enzyme is comprised of CI glucanotransferase (TtCITase) at the N-terminal region and long isomaltooligosaccharide-forming enzyme (TtTGase) at the C-terminal region connected by carbohydrate-binding module family 35 (CBM, TtCBM). Three truncated mutants of CI-forming enzymes were successfully produced in Corynebacterium glutamicum, a food-grade host system, and their biochemical properties were characterized. The enzymes had optimum at pH 6.0 and pH-stability (5.0-12.0). Three enzymes had optimum temperature over 55 °C and they maintained 80% activity at 55 °C for 2 h, 12 h, and 18 h, respectively. Enzymes without CBM showed weaker allosteric behavior than those of other enzymes, which suggests the important role of CBM in allosteric behavior. However, CBM bearing enzymes showed high production of CIs with various degree of polymerization. These enzymes have potential application as the encapsulating material for insoluble pharmaceutical biomaterials.

Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.

Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x-ray crystallography. The tetrameric structure of C295A-SADH soaked with NADP+ and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with an Rfree of 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys-37, NE2 of His-59, and OD2 of Asp-150. The nicotinamide ring of NADP is hydrogen-bonded to the N of Ala-295 and the O of Val-265 and Gly-293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser-39, the 3'-OH of NADP, and ND1 of His-42. The structure of I86A-SADH soaked with 2-pentanol and NADP+ contains (R)-2-pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A-SADH soaked with 3-methylcyclohexanol and NADP+ has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu-60 in the other axial position of a trigonal bipyramidal complex. One site has 3-methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu-60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu-60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu-60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium.

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.

The monofunctional CO dehydrogenase CooS is essential for growth of Thermoanaerobacter kivui on carbon monoxide.

Thermoanaerobacter kivui is a thermophilic acetogen that can grow on carbon monoxide as sole carbon and energy source. To identify the gene(s) involved in CO oxidation, the genome sequence was analyzed. Two genes potentially encoding CO dehydrogenases were identified. One, cooS, potentially encodes a monofunctional CO dehydrogenase, whereas another, acsA, potentially encodes the CODH component of the CODH/ACS complex. Both genes were cloned, a His-tag encoding sequence was added, and the proteins were produced from a plasmid in T. kivui. His-AcsA copurified by affinity chromatography with AcsB, the acetyl-CoA synthase of the CO dehydrogenase/acetyl CoA synthase complex. His-CooS copurified with CooF1, a small iron-sulfur center containing protein likely involved in electron transport. Both protein complexes had CO:ferredoxin oxidoreductase as well as CO:methyl viologen oxidoreductase activity, but the activity of CooSF1 was 15-times and 231-times lower, respectively. To underline the importance of CooS, the gene was deleted in the CO-adapted strain. Interestingly, the ∆cooS deletion mutant did not grow on CO anymore. These experiments clearly demonstrated that CooS is essential for growth of T. kivui on CO. This is in line with the hypothesis that CooS is the CO-oxidizing enzyme in cells growing on CO.

Reversible or Irreversible Catalysis of H+/H2 Conversion by FeFe Hydrogenases.

Studies of molecular catalysts traditionally aim at understanding how a certain mechanism allows the reaction to be fast. A distinct question, which has only recently received attention in the case of bidirectional molecular catalysts, is how much thermodynamic driving force is required to achieve fast catalysis in either direction of the reaction. "Reversible" catalysts are bidirectional catalysts that work either way in response to even a small departure from equilibrium and thus do not waste input free energy as heat; conversely, "irreversible" catalysts require a large driving force to proceed at an appreciable rate [Fourmond et al. Nat. Rev. Chem. 2021, 5, 348-360]. Numerous mechanistic rationales for these contrasting behaviors have been proposed. To understand the determinants of catalytic (ir)reversibility, we examined the steady-state, direct electron transfer voltammetry of a particular FeFe hydrogenase, from Thermoanaerobacter mathranii, which is very unusual in that it irreversibly catalyzes H2 oxidation and production: a large overpotential is required for the reaction to proceed in either direction [Land et al. Chem. Sci. 2020, 11, 12789-12801]. In contrast to previous hypotheses, we demonstrate that in this particular enzyme catalytic irreversibility can be explained without invoking slow interfacial electron transfer or variations in the mechanism: the observed kinetics is fully consistent with the same catalytic pathway being used in both directions of the reaction.