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The small heat-shock protein (sHSP) from the archaea Methanocaldococcus jannaschii , MjsHSP16.5, functions as a broad substrate ATP-independent holding chaperone protecting misfolded proteins from aggregation under stress conditions. This protein is the first sHSP characterized by X-ray crystallography, thereby contributing significantly to our understanding of sHSPs. However, despite numerous studies assessing its functions and structures, the precise arrangement of the N-terminal domains (NTDs) within this sHSP cage remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of MjsHSP16.5 at 2.49-Å resolution. The subunits of MjsHSP16.5 in the cryo-EM structure exhibit lesser compaction compared to their counterparts in the crystal structure. This structural feature holds particular significance in relation to the biophysical properties of MjsHSP16.5, suggesting a close resemblance to this sHSP native state. Additionally, our cryo-EM structure unveils the density of residues 24–33 within the NTD of MjsHSP16.5, a feature that typically remains invisible in the majority of its crystal structures. Notably, these residues show a propensity to adopt a β-strand conformation and engage in antiparallel interactions with strand β1, both intra- and inter-subunit modes. These structural insights are corroborated by structural predictions, disulfide bond cross-linking studies of Cys-substitution mutants, and protein disaggregation assays. A comprehensive understanding of the structural features of MjsHSP16.5 expectedly holds the potential to inspire a wide range of interdisciplinary applications, owing to the renowned versatility of this sHSP as a nanoscale protein platform. [ABSTRACT FROM AUTHOR]

Alpha amylases catalyse the hydrolysis of α-1, 4-glycosidic bonds in starch, yielding glucose, maltose, dextrin, and short oligosaccharides, vital to various industrial processes. Structural and functional insights on α-amylase from Methanocaldococcus jannaschii were computationally explored to evaluate a catalytic domain and its fusion with a small ubiquitin-like modifier (SUMO). The recombinant proteins' production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene (MjAFG), catalytic domain (MjAD) and fusion enzymes (S-MjAD) were thoroughly analysed in this comparative study. The MjAD and S-MjAD showed 2-fold and 2.5-fold higher specific activities (μmol min−1 mg −1) than MjAFG at 95 °C at pH 6.0. Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the MjAFG enzyme. The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. The chimeric fusion of amylases with thermostable α-glucosidases makes it a potential candidate for the starch degrading processes. • The structure and function features of α-amylase in Methanocaldococcus jannaschii have been comprehensively analyzed. Protein engineering technologies have been used to improve its catalytic activities, thermostability stability and solubility-related parameters • The recombinant protein production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene, catalytic domain and fusion enzymes were thoroughly explored in this comparative study. • The catalytic domain and fusion enzyme showed 2-fold and 2.5-fold higher specific activities (μmol min-1 mg -1) than the native enzyme respectively. • Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the native full enzyme. • The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. • HPLC and LC-MS spectrometry analysis were performed for a better understanding of high readership interest. • The thermostability of this enzyme at 100-120 oC makes it a potential candidate for different industrial processes. [ABSTRACT FROM AUTHOR]

Phylogenetically deeply rooted methanogens belonging to the genus of Methanocaldococcus living in deep-sea hydrothermal vents derive energy exclusively from hydrogenotrophic methanogenesis, one of the oldest respiratory metabolisms on Earth. These hyperthermophilic, autotrophic archaea synthesize their biomolecules from inorganic substrates and perform high temperature biocatalysis producing methane, a valuable fuel and potent greenhouse gas. The information processing and stress response systems of archaea are highly homologous to those of the eukaryotes. For this broad relevance, Methanocaldococcus jannaschii, the first hyperthermophilic chemolithotrophic organism that was isolated from a deep-sea hydrothermal vent, was also the first archaeon and third organism for which the whole genome sequence was determined. The research that followed uncovered numerous novel information in multiple fields, including those described above. M. jannaschii was found to carry ancient redox control systems, precursors of dissimilatory sulfate reduction enzymes, and a eukaryotic-like protein translocation system. It provided a platform for structural genomics and tools for incorporating unnatural amino acids into proteins. However, the assignments of in vivo relevance to these findings or interrogations of unknown aspects of M. jannaschii through genetic manipulations remained out of reach, as the organism was genetically intractable. This report presents tools and methods that remove this block. It is now possible to knockout or modify a gene in M. jannaschii and genetically fuse a gene with an affinity tag sequence, thereby allowing facile isolation of a protein with M. jannaschii-specific attributes. These tools have helped to genetically validate the role of a novel coenzyme F420-dependent sulfite reductase in conferring resistance to sulfite in M. jannaschii and to demonstrate that the organism possesses a deazaflavin-dependent system for neutralizing oxygen.

Phylogenetically deeply rooted methanogens belonging to the genus of Methanocaldococcus living in deep-sea hydrothermal vents derive energy exclusively from hydrogenotrophic methanogenesis, one of the oldest respiratory metabolisms on Earth. These hyperthermophilic, autotrophic archaea synthesize their biomolecules from inorganic substrates and perform high temperature biocatalysis producing methane, a valuable fuel and potent greenhouse gas. The information processing and stress response systems of archaea are highly homologous to those of the eukaryotes. For this broad relevance, Methanocaldococcus jannaschii , the first hyperthermophilic chemolithotrophic organism that was isolated from a deep-sea hydrothermal vent, was also the first archaeon and third organism for which the whole genome sequence was determined. The research that followed uncovered numerous novel information in multiple fields, including those described above. M. jannaschii was found to carry ancient redox control systems, precursors of dissimilatory sulfate reduction enzymes, and a eukaryotic-like protein translocation system. It provided a platform for structural genomics and tools for incorporating unnatural amino acids into proteins. However, the assignments of in vivo relevance to these findings or interrogations of unknown aspects of M. jannaschii through genetic manipulations remained out of reach, as the organism was genetically intractable. This report presents tools and methods that remove this block. It is now possible to knockout or modify a gene in M. jannaschii and genetically fuse a gene with an affinity tag sequence, thereby allowing facile isolation of a protein with M. jannaschii -specific attributes. These tools have helped to genetically validate the role of a novel coenzyme F420-dependent sulfite reductase in conferring resistance to sulfite in M. jannaschii and to demonstrate that the organism possesses a deazaflavin-dependent system for neutralizing oxygen. [ABSTRACT FROM AUTHOR]

Abstract The use of modular domains in proteins affords nature a simple route to the diversification of protein function, but co-evolution between domains can complicate large-scale functional annotation. The LeuA dimer regulatory domain is primarily responsible for allosteric feedback inhibition of the enzymes isopropylmalate synthase (IPMS) and citramalate synthase (CMS). In addition to this regulatory role, presence of the domain may also affect substrate selectivity in certain members of the enzyme family. To assess the role of the LeuA dimer regulatory domain in substrate selectivity, truncated versions of IPMS and CMS from Methanococcus jannaschii (MjIPMS and MjCMS, respectively) have been created that lack the LeuA dimer regulatory domain. In the case of MjIPMS, loss of the regulatory domain does not affect substrate selectivity, consistent with previous reports identifying conserved active site residues that play this role. Loss of the regulatory domain in MjCMS, however, results in increased functional promiscuity. Both truncated enzymes exhibit a shift in quaternary structure from tetrameric to monomeric forms as judged by size-exclusion chromatography. Kinetic isotope effects reveal that loss of the regulatory domain results in unique effects on catalysis with chemistry becoming more rate-determining in MjIPMS and less rate-determining in MjCMS. Finally, substitution of conserved active site residues in the promiscuous truncated MjCMS affect substrate selectivity while identical substitutions cause no changes in the wild-type enzyme. Overall, the data predicts a more complex role for the LeuA dimer regulatory domain in substrate selectivity through catalytic modulations rather than selectivity through differential binding as a result of extensive co-evolution between the catalytic and regulatory domains. Highlights • Removal of the allosteric regulatory domain in MjCMS alters substrate selectivity. • Catalysis by MjIPMS and MjCMS are differentially affected by loss of the domain. • Truncation causes changes in quaternary structure in both enzymes. • Active site substitutions of truncated MjCMS further alter substrate selectivity. [ABSTRACT FROM AUTHOR]

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called "posttranslational proofreading" to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code. [ABSTRACT FROM AUTHOR]

2-Methylsuccinic acid (2-MSA) is a C5 branched-chain dicarboxylate that serves as an attractive synthon for the synthesis of polymers with extensive applications in coatings, cosmetic solvents and bioplastics. However, the lack of natural pathways for 2-MSA biosynthesis has limited its application as a promising bio-replacement. Herein, we conceived a non-natural three-step biosynthetic route for 2-MSA, via employing the citramalate pathway in combination with enoate reductase-mediated bioreduction of the pathway intermediate citraconate. First, over-expression of codon-optimized citramalate synthase variant CimA* from Methanococcus jannaschii , endogenous isopropylmalate isomerase EcLeuCD and enoate reductase YqjM from Bacillus subtilis allowed the production of 2-MSA in Escherichia coli for the first time, with a titer of 0.35 g/L in shake flask experiments. Subsequent screening of YqjM-like enoate reductases of different bacterial origins enabled identification and characterization of a new NAD(P)H-dependent enoate reductase KpnER from Klebsiella pneumoniae , which exhibited higher activity towards citraconate than YqjM. Incorporation of KpnER into the 2-MSA biosynthetic pathway led to 2-MSA production improvement to a titer of 0.96 g/L in aerobic condition. Subsequent optimizations including cofactor regeneration, microaerobic cultivation and host strain engineering, boosted 2-MSA titer to 3.61 g/L with a molar yield of 0.36 in shake flask experiments. This work established a promising platform for 2-MSA bioproduction, which enabled the highest titer of 2-MSA production in microbial hosts so far. [ABSTRACT FROM AUTHOR]

Background Adding new amino acids to the set of building blocks for protein synthesis expands the scope of protein engineering, and orthogonal pairs of tRNA and aminoacyl-tRNA synthetase have been developed for incorporating unnatural amino acids (UAAs) into proteins. While diverse systems have been developed to incorporate UAAs in response to the amber codon, less research has been focused on four-base codons despites their advantages. In this study, we report an efficient method to incorporate UAA in response to an AGGA codon in Escherichia coli . Results The Methanococcus jannaschii tyrosyl-tRNA synthetase-tRNA CUA (MjTyrRS-MjtRNA CUA ) orthogonal pair has been engineered to incorporate diverse UAAs in response to the amber codon. To apply the engineered MjTyrRS enzymes for UAAs to a four-base codon suppression, we developed an MjTyrRS-MjtRNA UCCU pair system that enabled incorporation of UAAs in response to the AGGA codon in E. coli . Using this system, we demonstrated that several UAAs could be incorporated quantitatively in the AGGA site. In addition, multiple AGGA codons were successfully suppressed in an E. coli strain when the endogenous tRNA CCU Arg gene was knocked out. Conclusion An efficient system was developed for the incorporation of UAAs in response to the AGGA four-base codon in E. coli , and the method was successfully demonstrated for several UAAs and for multiple AGGA sites. General significance The developed system can expand the repertoire of protein engineering tools based on amino acid analogues in combination with other UAA incorporation methods. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O’Donoghue. [ABSTRACT FROM AUTHOR]

The CRISPR-associated protein 6, Cas6 protein, is an endoribonuclease that cleaves precursor CRISPR RNAs within the repeat sequence to release specific invader-targeting RNAs. Cas6 protein can recognize different sequences by their specific scaffold. To investigate its binding mode, we purified and crystallized a His-tagged Cas6 protein from Methanocaldococcus jannaschii (MjCas6) using the sitting-drop vapor-diffusion method. The crystals diffracted to a resolution of 1.85 Å and belonged to monoclinic space group C2, with unit-cell parameters a = 200.84 Å, b = 85.26 Å, c = 100.06 Å, β= 118.47°. The crystals of MjCas6 contain four molecules in the asymmetric unit. The protein fold is similar to the other Cas6 homologues, such as Pyrococcus furiosus Cas6, suggesting functional similarity. Moreover, in the C2 crystal the MjCas6 monomers formed a tandem array, which we hypothesize to possibly correlate with repetitive RNA precursors. [ABSTRACT FROM AUTHOR]

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1–2 μM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization. [ABSTRACT FROM AUTHOR]

Citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. We compared citramalate and acetate accumulation from glycerol using Escherichia coli strains expressing a modified citramalate synthase gene cimA from Methanococcus jannaschii. These studies revealed that gltA coding citrate synthase, leuC coding 3-isopropylmalate dehydratase, and acetate pathway genes play important roles in elevating citramalate and minimizing acetate formation. Controlled 1.0 L batch experiments confirmed that deletions in all three acetate-production genes ( poxB, ackA, and pta) were necessary to reduce acetate formation to less than 1 g/L during citramalate production from 30 g/L glycerol. Fed-batch processes using MEC568/pZE12- cimA ( gltA leuC ackA- pta poxB) generated over 31 g/L citramalate and less than 2 g/L acetate from either purified or crude glycerol at yields exceeding 0.50 g citramalate/g glycerol in 132 h. These results hold promise for the viable formation of citramalate from unrefined glycerol. [ABSTRACT FROM AUTHOR]

Bacterial nuclease RecJ, which exists in almost all bacterial species, specifically degrades single-stranded (ss) DNA in the 5' to 3' direction. Some archaeal phyla, except Crenarchaea, also encode RecJ homologs. Compared with bacterial RecJ, archaeal RecJ exhibits a largely different amino acid sequence and domain organization. Archaeal RecJs from Thermococcus kodakarensis and Pyrococcus furiosus show 5'→3' exonuclease activity on ssDNA. Interestingly, more than one RecJ exists in some Euryarchaeota classes, such as Methanomicrobia, Methanococci, Methanomicrobia, Methanobacteria, and Archaeoglobi. Here we report the biochemical characterization of two RecJs from Methanocaldococcus jannaschii, the long RecJ1 (MJ0977) and short RecJ2 (MJ0831) to understand their enzymatic properties. RecJ1 is a 5'→3' exonuclease with a preference to ssDNA; however, RecJ2 is a 3'→5' exonuclease with a preference to ssRNA. The 5' terminal phosphate promotes RecJ1 activity, but the 3' terminal phosphate inhibits RecJ2 nuclease. Go-Ichi-Ni-San (GINS) complex does not interact with two RecJs and does not promote their nuclease activities. Finally, we discuss the diversity, function, and molecular evolution of RecJ in archaeal taxonomy. Our analyses provide insight into the function and evolution of conserved archaeal RecJ/eukaryotic Cdc45 protein. [ABSTRACT FROM AUTHOR]

The gene that codes for the putative dihydroorotase in the hyperthermophilic archaeon Methanococcus jannaschii was subcloned in pET-21a and expressed in Escherichia coli. A purification protocol was devised. The purity of the protein was evaluated by SDS-PAGE and the protein was confirmed by sequencing using LC-MS. The calculated molecular mass is 48104 Da. SEC-LS suggested that the protein is a monomer in solution. ICP-MS showed that there are two Zn ions per monomer. Kinetic analysis of the recombinant protein gave hyperbolic kinetics with V = 12.2 µmol/min/mg and K = 0.14 mM at 25 °C. Furthermore the activity of the protein increased with temperature consistent with the hyperthermophilic nature of the organism. A homology model was constructed using the mesophilic Bacillus anthracis protein as the template. Residues known to be critical for Zn and substrate binding were conserved. The activity of the enzyme at 85 and 90 °C was found to be relatively constant over 160 min and this correlates with the temperature of optimal growth of the organism of 85 °C. The amino acid sequences and structures of the two proteins were compared and this gave insight into some of the factors that may confer thermostability-more Lys and Ile, fewer Ala, Thr, Gln and Gly residues, and shorter N- and C-termini. Additional and better insight into the thermostabilization strategies adopted by this enzyme will be provided when its crystal structure is determined. [ABSTRACT FROM AUTHOR]

In Methanocaldococcus jannaschii, methylglyoxal (MG) is required for aromatic amino acid biosynthesis. Previously, the reduction of MG to lactaldehyde in Methanocaldococcus jannaschii cell extracts using either NADPH or F420H2 was demonstrated; however, the enzyme responsible was not identified. Using NADPH as the reductant, the unknown enzyme was purified from cell extracts of Methanocaldococcus jannaschii and determined to be the F420-dependent N5 ,N10-methylenetetrahydromethanopterin reductase (Mer). Here, we report that the recombinantly overexpressed Mer is able to use NADPH and MG ( KM of 1.6 and 1.0 m m, respectively) to produce lactaldehyde. Additionally, Mer does not catalyze the reduction of MG to lactaldehyde in the presence of reduced Fo, the precursor of F420. [ABSTRACT FROM AUTHOR]

The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus. [ABSTRACT FROM AUTHOR]

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschiiNCX)system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ionbound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations. [ABSTRACT FROM AUTHOR]

The crystal structure of a hypothetical protein MJ0366, derived from Methanocaldococcus jannaschii was solved at 1.9 Å resolution using synchrotron radiation. MJ0366 was crystallized as a monomer and has knot structural arrangement. Intriguingly, the solved structure consists of novel ‘KNOT’ fold conformation. The 3 1 trefoil knot was observed in the structure. The N-terminal and C-terminal ends did not participate in knot formation. [ABSTRACT FROM AUTHOR]

Greigite, a ferrimagnetic iron sulfide Fe(II)Fe(III) 2 S 4 , is thought to have played an essential role in chemical evolution leading to the origin of life. Greigite contains a [4Fe–4S] cluster-like structure and has been synthesized in the laboratory by liquid-state reactions. However, it is unclear how greigite can be synthesized in nature. Herein, we show that greigite is synthesized by the solid–gas reaction of Fe(III)-oxide-hydroxides and H 2 S. We discovered that the hyperthermophilic hydrogenotrophic methanogen Methanocaldococcus jannaschii reduced elemental sulfur, and the resulting sulfide generated greigite from hematite. The time course and pH dependence of the reaction respectively indicated the involvement of amorphous FeS and H 2 S as reaction intermediates. An abiotic solid–gas reaction of hematite and H 2 S (g) under strictly anaerobic conditions was developed. The solid–gas reaction fully converted hematite to greigite/pyrite at 40–120 °C within 12 h and was unaffected by the bulk gas phase. Similar abiotic reactions occurred, but relatively slowly, with aqueous H 2 S in acidulous liquids using hematite, magnetite, or amorphous FeO(OH) as starting materials, suggesting that greigite was extensively produced in the Hadean Eon as these Fe(III)-oxide-hydroxides were shown to be present or routinely produced during that era. Surprisingly, the obtained greigite induced methanogenesis and growth of hydrogenotrophic methanogens, suggesting that the external greigite crystals enhanced reactions that would otherwise require enzymes, such as [4Fe–4S] cluster-harboring membrane-bound hydrogenases. These data suggested that the greigite produced by the solid–gas and solid–dissolved gas reactions was bioactive. [ABSTRACT FROM AUTHOR]

Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation. [ABSTRACT FROM AUTHOR]

In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannaschii, we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA) and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M. jannaschii. We show that HMBA can be biosynthesized from malate semialdehyde and hydrogen sulfide, likely using a mechanism similar to that proposed for coenzyme M, coenzyme B and homocysteine biosynthesis in methanogens, where an aldehyde is converted to a thiol. The L-sulfolactate dehydrogenase, derived from the MJ1425 gene, is shown to catalyze the NAD-dependent oxidation of HMBA to MOB. Finally, we demonstrate that HMBA can be used as a biosynthetic precursor to MPA in M. jannaschii cell extracts. This proposed pathway may contribute to the wide occurrence of MPA in marine environments and indicates that MPA must serve some important function in M. jannaschii. [ABSTRACT FROM AUTHOR]

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion. [ABSTRACT FROM AUTHOR]

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5′-monophosphate (GTP) and aspartate to convert inosine 5′-monophosphate (IMP) into succinyl adenosine 5′-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5′-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis. [ABSTRACT FROM AUTHOR]

ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 ( MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here, Methanococcus jannaschii MR- ATPγS- DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity. [ABSTRACT FROM AUTHOR]

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 109-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment. [ABSTRACT FROM AUTHOR]

Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 in two complementary states. The inward-open state was obtained by x-ray crystallography in the presence of sodium at pH 8, where the transporter is highly active. The outward-open state was obtained by electron crystallography without sodium at pH 4, where MjNhaP1 is inactive. Comparison of both structures reveals a 7° tilt of the 6 helix bundle. 22Na+ uptake measurements indicate non-cooperative transport with an activity maximum at pH 7.5. We conclude that binding of a Na+ ion from the outside induces helix movements that close the extracellular cavity, open the cytoplasmic funnel, and result in a ∼5 Å vertical relocation of the ion binding site to release the substrate ion into the cytoplasm.

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time. [ABSTRACT FROM AUTHOR]

CorA channels are responsible for the uptake of essential magnesium ions by bacteria. X-ray crystal structures have been resolved for two full-length CorA channels, each in a non-conducting state with magnesium ions bound to the protein: These structures reveal a homo-pentameric quaternary structure with approximate 5-fold rotational symmetry about a central pore axis. We report the structure of the detergent solubilized Methanocaldococcus jannaschii CorA channel determined by Cryo-Electron Microscopy and Single Particle Averaging, supported by Small Angle X-ray Scattering and X-ray crystallography. This structure also shows a pentameric channel but with a highly asymmetric domain structure. The asymmetry of the domains includes differential separations between the trans-membrane segments, which reflects mechanical coupling of the cytoplasmic domain to the trans-membrane domain. This structure therefore reveals an important aspect of the gating mechanism of CorA channels by providing an indication of how the absence of magnesium ions leads to major structural changes. [ABSTRACT FROM AUTHOR]

Summary Cells possess specialized machinery to direct the insertion of membrane proteins into the lipid bilayer. In bacteria, the essential protein YidC inserts certain proteins into the plasma membrane, and eukaryotic orthologs are present in the mitochondrial inner membrane and the chloroplast thylakoid membrane. The existence of homologous insertases in archaea has been proposed based on phylogenetic analysis. However, limited sequence identity, distinct architecture, and the absence of experimental data have made this assignment ambiguous. Here we describe the 3.5-Å crystal structure of an archaeal DUF106 protein from Methanocaldococcus jannaschii ( Mj 0480), revealing a lipid-exposed hydrophilic surface presented by a conserved YidC-like fold. Functional analysis reveals selective binding of Mj 0480 to ribosomes displaying a stalled YidC substrate, and a direct interaction between the buried hydrophilic surface of Mj 0480 and the nascent chain. These data provide direct experimental evidence that the archaeal DUF106 proteins are YidC/Oxa1/Alb3-like insertases of the archaeal plasma membrane. [ABSTRACT FROM AUTHOR]

All methanofuran structural variants contain a basic core structure of 4-[N-(γ-L-glutamyl)-p-(β-aminoethyl)phenoxymethyl](aminomethyl)furan (APMF-Glu) but have different side chains depending on the source organism. Recently, we identified four genes (MfnA, MfnB, MfnC, and MfnD) that are responsible for the biosynthesis of the methanofuran precursor γ-glutamyltyramine and 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P) from tyrosine, glutamate, glyceraldehyde-3-P, and alanine in Methanocaldococcus jannaschii. How γ-glutamyltyramine and F1-P couple together to form the core structure of methanofuran was previously unknown. Here, we report the identification of two enzymes encoded by the genes mj0458 and mj0840 that catalyze the formation of F1-PP from ATP and F1-P and the condensation of F1-PP with γ-glutamyltyramine, respectively, to form APMF-Glu. We have annotated these enzymes as MfnE and MfnF, respectively, representing the fifth and sixth enzymes in the methanofuran biosynthetic pathway to be identified. Although MfnE was previously reported as an archaeal adenylate kinase, our present results show that MfnE is a promiscuous enzyme and that its possible physiological role is to produce F1-PP. Unlike other enzymes catalyzing coupling reactions involving pyrophosphate as the leaving group, MfnF exhibits a distinctive α/β two-layer sandwich structure. By comparing MfnF with thiamine synthase and dihydropteroate synthase, a substitution nucleophilic unimolecular (SN-1) reaction mechanism is proposed for MfnF. With the identification of MfnE and MfnF, the biosynthetic pathway for the methanofuran core structure APMF-Glu is complete. [ABSTRACT FROM AUTHOR]

Prefoldins ( PFDs) are molecular chaperones with a distinctive jellyfish-shape that have a general role in de novo protein folding in Archaea and in the biogenesis of cytoskeleton proteins in eukaryotes. In general, PFDs are hetero-hexameric protein assemblies consisting of two α and four β subunits. However, a PFD variant called gamma-prefoldin (γ PFD), isolated from the hyperthermophilic archaeon Methanocaldococcus jannaschii, exhibits a unique filamentous structure that is composed of hundreds of monomeric subunits. In this study, we investigated the relationship between the morphology of the γ PFD filament and its ability to prevent protein aggregation. A chaperone assay demonstrated that γ PFD must be in a filamentous assembly for functional activity and the distal regions of the coiled-coils are required for binding of non-native proteins. Molecular dynamic simulations were used to model the interactions between in silico thermally denatured protein substrates and the coiled-coils of a γ PFD filament. During molecular dynamic simulations at 300 and 353 K, each coiled-coil was highly flexible, enabling it to widen the central cavity of the filament to potentially capture various non-native proteins. Docking molecular dynamic simulations of γ PFD filaments with unfolded citrate synthase or insulin showed a size-dependence between the substrate and the number of interacting coiled-coils. To confirm this observation, we generated filaments containing specific numbers of subunits, and showed that between six and eight γ PFD subunits are required for chaperone activity to prevent citrate synthase from thermal aggregation. These results provide insights into structure-function relationships of oligomeric chaperones and illuminate the potential role of γ PFD in its native environment. [ABSTRACT FROM AUTHOR]