Differences in regulation mechanisms of glutamine synthetases from methanogenic archaea unveiled by structural investigations.

Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50'-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Structural and biochemical elucidation of class I hybrid cluster protein natively extracted from a marine methanogenic archaeon.

Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from Desulfovibrio species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from Methanococcales and many Bacteria corroborates this similarity. Indeed, Methanococcales HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in M. thermolithotrophicus, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.

A Structural View of Alkyl-Coenzyme M Reductases, the First Step of Alkane Anaerobic Oxidation Catalyzed by Archaea.

Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications. Archaea are proposed to degrade short- and long-chain alkanes to CO2 by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural knowledge of alkyl-CoM reductases and offers fresh ideas about the expected mechanistic and chemical differences among members of this broad family. We conclude with the challenges of the investigation of these particular enzymes, which might one day generate biofuels for our modern society.

Crystal structure of a key enzyme for anaerobic ethane activation.

Ethane, the second most abundant hydrocarbon gas in the seafloor, is efficiently oxidized by anaerobic archaea in syntrophy with sulfate-reducing bacteria. Here, we report the 0.99-angstrom-resolution structure of the proposed ethane-activating enzyme and describe the specific traits that distinguish it from methane-generating and -consuming methyl-coenzyme M reductases. The widened catalytic chamber, harboring a dimethylated nickel-containing F430 cofactor, would adapt the chemistry of methyl-coenzyme M reductases for a two-carbon substrate. A sulfur from methionine replaces the oxygen from a canonical glutamine as the nickel lower-axial ligand, a feature conserved in thermophilic ethanotrophs. Specific loop extensions, a four-helix bundle dilatation, and posttranslational methylations result in the formation of a 33-angstrom-long hydrophobic tunnel, which guides the ethane to the buried active site as confirmed with xenon pressurization experiments.

Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum.

Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2ß2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.

CO2-Fixation Strategies in Energy Extremophiles: What Can We Learn From Acetogens?

Domestication of CO2-fixation became a worldwide priority enhanced by the will to convert this greenhouse gas into fuels and valuable chemicals. Because of its high stability, CO2-activation/fixation represents a true challenge for chemists. Autotrophic microbial communities, however, perform these reactions under standard temperature and pressure. Recent discoveries shine light on autotrophic acetogenic bacteria and hydrogenotrophic methanogens, as these anaerobes use a particularly efficient CO2-capture system to fulfill their carbon and energy needs. While other autotrophs assimilate CO2 via carboxylation followed by a reduction, acetogens and methanogens do the opposite. They first generate formate and CO by CO2-reduction, which are subsequently fixed to funnel the carbon toward their central metabolism. Yet their CO2-reduction pathways, with acetate or methane as end-products, constrain them to thrive at the "thermodynamic limits of Life". Despite this energy restriction acetogens and methanogens are growing at unexpected fast rates. To overcome the thermodynamic barrier of CO2-reduction they apply different ingenious chemical tricks such as the use of flavin-based electron-bifurcation or coupled reactions. This mini-review summarizes the current knowledge gathered on the CO2-fixation strategies among acetogens. While extensive biochemical characterization of the acetogenic formate-generating machineries has been done, there is no structural data available. Based on their shared mechanistic similarities, we apply the structural information obtained from hydrogenotrophic methanogens to highlight common features, as well as the specific differences of their CO2-fixation systems. We discuss the consequences of their CO2-reduction strategies on the evolution of Life, their wide distribution and their impact in biotechnological applications.

The Shewanella genus: ubiquitous organisms sustaining and preserving aquatic ecosystems.

The Gram-negative Shewanella bacterial genus currently includes about 70 species of mostly aquatic γ--proteobacteria, which were isolated around the globe in a multitude of environments such as surface freshwater and the deepest marine trenches. Their survival in such a wide range of ecological niches is due to their impressive physiological and respiratory versatility. Some strains are among the organisms with the highest number of respiratory systems, depending on a complex and rich metabolic network. Implicated in the recycling of organic and inorganic matter, they are important components of organism-rich oxic/anoxic interfaces, but they also belong to the microflora of a broad group of eukaryotes from metazoans to green algae. Examples of long-term biological interactions like mutualism or pathogeny have been described, although molecular determinants of such symbioses are still poorly understood. Some of these bacteria are key organisms for various biotechnological applications, especially the bioremediation of hydrocarbons and metallic pollutants. The natural ability of these prokaryotes to thrive and detoxify deleterious compounds explains their use in wastewater treatment, their use in energy generation by microbial fuel cells and their importance for resilience of aquatic ecosystems.

Shewanella decolorationis LDS1 Chromate Resistance.

The genus Shewanella is well known for its genetic diversity, its outstanding respiratory capacity, and its high potential for bioremediation. Here, a novel strain isolated from sediments of the Indian Ocean was characterized. A 16S rRNA analysis indicated that it belongs to the species Shewanella decolorationis It was named Shewanella decolorationis LDS1. This strain presented an unusual ability to grow efficiently at temperatures from 24°C to 40°C without apparent modifications of its metabolism, as shown by testing respiratory activities or carbon assimilation, and in a wide range of salt concentrations. Moreover, S. decolorationis LDS1 tolerates high chromate concentrations. Indeed, it was able to grow in the presence of 4 mM chromate at 28°C and 3 mM chromate at 40°C. Interestingly, whatever the temperature, when the culture reached the stationary phase, the strain reduced the chromate present in the growth medium. In addition, S. decolorationis LDS1 degrades different toxic dyes, including anthraquinone, triarylmethane, and azo dyes. Thus, compared to Shewanella oneidensis, this strain presented better capacity to cope with various abiotic stresses, particularly at high temperatures. The analysis of genome sequence preliminary data indicated that, in contrast to S. oneidensis and S. decolorationis S12, S. decolorationis LDS1 possesses the phosphorothioate modification machinery that has been described as participating in survival against various abiotic stresses by protecting DNA. We demonstrate that its heterologous production in S. oneidensis allows it to resist higher concentrations of chromate.IMPORTANCEShewanella species have long been described as interesting microorganisms in regard to their ability to reduce many organic and inorganic compounds, including metals. However, members of the Shewanella genus are often depicted as cold-water microorganisms, although their optimal growth temperature usually ranges from 25 to 28°C under laboratory growth conditions. Shewanella decolorationis LDS1 is highly attractive, since its metabolism allows it to develop efficiently at temperatures from 24 to 40°C, conserving its ability to respire alternative substrates and to reduce toxic compounds such as chromate or toxic dyes. Our results clearly indicate that this novel strain has the potential to be a powerful tool for bioremediation and unveil one of the mechanisms involved in its chromate resistance.

Draft Genome Sequence of Shewanella algidipiscicola H1, a Highly Chromate-Resistant Strain Isolated from Mediterranean Marine Sediments.

The ability of different Shewanella spp. to convert heavy metals and toxic substances into less toxic products by using them as electron acceptors has led to their use in environmental clean-up strategies. We present here the draft genome sequence of Shewanella algidipiscicola H1, a strain resistant to high concentrations of chromates.

Small membranous proteins of the TorE/NapE family, crutches for cognate respiratory systems in Proteobacteria.

In this report, we investigate small proteins involved in bacterial alternative respiratory systems that improve the enzymatic efficiency through better anchorage and multimerization of membrane components. Using the small protein TorE of the respiratory TMAO reductase system as a model, we discovered that TorE is part of a subfamily of small proteins that are present in proteobacteria in which they play a similar role for bacterial respiratory systems. We reveal by microscopy that, in Shewanella oneidensis MR1, alternative respiratory systems are evenly distributed in the membrane contrary to what has been described for Escherichia coli. Thus, the better efficiency of the respiratory systems observed in the presence of the small proteins is not due to a specific localization in the membrane, but rather to the formation of membranous complexes formed by TorE homologs with their c-type cytochrome partner protein. By an in vivo approach combining Clear Native electrophoresis and fluorescent translational fusions, we determined the 4:4 stoichiometry of the complexes. In addition, mild solubilization of the cytochrome indicates that the presence of the small protein reinforces its anchoring to the membrane. Therefore, assembly of the complex induced by this small protein improves the efficiency of the respiratory system.

Chaperones in maturation of molybdoenzymes: Why specific is better than general?

Molybdoenzymes play essential functions in living organisms and, as a result, in various geochemical cycles. It is thus crucial to understand how these complex proteins become highly efficient enzymes able to perform a wide range of catalytic activities. It has been established that specific chaperones are involved during their maturation process. Here, we raise the question of the involvement of general chaperones acting in concert with dedicated chaperones or not.

Efficient respiration on TMAO requires TorD and TorE auxiliary proteins in Shewanella oneidensis.

Respiration on trimethylamine oxide (TMAO) allows bacterial survival under anoxia. In Shewanella oneidensis, Tor is the system involved in TMAO respiration and it is encoded by the torECAD operon. The torA and torC genes encode TorA terminal reductase and the TorC c-type cytochrome, respectively. Sequence analysis suggests that TorD is the putative specific chaperone of TorA, whereas TorE is of unknown function. The purpose of this study was to understand whether TorD and TorE are two accessory proteins that affect the efficiency of the Tor system by chaperoning TorA terminal reductase. Moreover, by deleting each gene, we established that the absence of TorD drastically affects the stability of TorA, while the absence of TorE does not affect TorA stability or activity. Since TMAO reduction was affected in the ΔtorE mutant, TorE could be an additional component of the TorC-TorA electron transfer chain during bacterial respiration. Finally, a fitness experiment indicated that the presence of TorE, as expected, confers a selective advantage in competitive environments.