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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]

Ribosomal protein L11 is an important part of the GTPase-associated centre in ribosomes of all organisms. L11 is a highly conserved two-domain ribosomal protein. The C-terminal domain of L11 is an RNA-binding domain that binds to a fragment of 23S rRNA and stabilizes its structure. The complex between L11 and 23S rRNA is involved in the GTPase activity of the translation elongation and release factors. Bacterial and archaeal L11-rRNA complexes are targets for peptide antibiotics of the thiazole class. To date, there is no complete structure of archaeal L11 owing to the mobility of the N-terminal domain of the protein. Here, the crystallization and X-ray analysis of the ribosomal protein L11 from Methanococcus jannaschii are reported. Crystals of the native protein and its selenomethionine derivative belonged to the orthorhombic space group I222 and were suitable for structural studies. Native and single-wavelength anomalous dispersion data sets have been collected and determination of the structure is in progress. [ABSTRACT FROM AUTHOR]

S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl- L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD+ and has Km values of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11mMfor inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. IMPORTANCE: In strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the biochemical reactions that were occurring when life originated. [ABSTRACT FROM AUTHOR]

In this work, we study dynamicalproperties of an extremophilicprotein, Initiation Factor 6 (IF6), produced by the archeabacterium Methanocaldococcus jannascii, which thrives close to deep-seahydrothermal vents where temperatures reach 80 °C and the pressureis up to 750 bar. Molecular dynamics simulations (MD) and quasi-elasticneutron scattering (QENS) measurements give new insights into thedynamical properties of this protein with respect to its eukaryoticand mesophilic homologue. Results obtained by MD are supported byQENS data and are interpreted within the framework of a fractionalBrownian dynamics model for the characterization of protein relaxationdynamics. IF6 from M. jannaschiiat high temperatureand pressure shares similar flexibility with its eukaryotic homologuefrom S. cerevisieaeunder ambient conditions. Thiswork shows for the first time, to our knowledge, that the very commonpattern of corresponding statesfor thermophilicprotein adaptation can be extended to thermo-barophilic proteins.A detailed analysis of dynamic properties and of local structuralfluctuations reveals a complex pattern for “corresponding”structural flexibilities. In particular, in the case of IF6, the latterseems to be strongly related to the entropic contribution given byan additional, C-terminal, 20 amino-acid tail which is evolutionaryconserved in all mesophilic IF6s. [ABSTRACT FROM AUTHOR]

Two-pore domain potassium (K) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K channels. Homologs of Arabidopsis K channels have been found in all higher plants sequenced so far. As with the K channels in mammals, plant K channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K channels are modulated by signaling molecules such as intracellular H and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K channels occurred in a prokaryote LCA. Conversely, we argue that the K LCA was most likely a eukaryote organism. Consideration of plant and animal K channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields. [ABSTRACT FROM AUTHOR]

Bacterial Hfq proteins are structural homologs of archaeal and eukaryotic Sm/Lsm proteins, which are characterized by a 5-stranded β-sheet and an N-terminal α-helix. Previously, it was shown that archaeal Lsm proteins (SmAP) could produce long fibrils spontaneously, in contrast to the Hfq from Escherichia coli that could form similar fibrils only after special treatment. The organization of these fibrils is significantly different, but the reason for the dissimilarity has not been found. In the present work, we studied the process of fibril formation by bacterial protein Hfq from Pseudomonas aeruginosa and archaeal protein SmAP from Methanococcus jannaschii. Both proteins have high homology with E. coli Hfq. We found that Hfq from P. aeruginosa could form fibrils after substitutions in the conserved Sm2 motif only. SmAP from M. jannaschii, like other archaeal Lsm proteins, form fibrils spontaneously. Despite differences in the fibril formation conditions, the architecture of both was similar to that described for E. coli Hfq. Therefore, universal nature of fibril architecture formed by Hfq proteins is suggested. [ABSTRACT FROM AUTHOR]

Understanding the mechanism by which polypeptide chains thread themselves into topologically knotted structures has emerged to be a challenging subject not least because of the additional complexity associated with the spontaneous and efficient knotting and folding events. While recent theoretical calculations have made significant progress in establishing the atomistic folding pathways for a number of knotted proteins, experimental data on the folding stabilities and kinetic pathways of knotted proteins has been sparse. Using MJ0366 from Methanocaldococcus jannaschii, the smallest knotted protein known to date, as a model system, we set out to systematically investigate its folding equilibrium, kinetics, and internal dynamics under native and chemically denatured states. NMR hydrogen-deuterium exchange analysis indicates that the knotted region is the most stable structural element within the novel fold. Additionally, 15N spin relaxation analysis reveals the presence of residual structures in urea-denatured MJ0366. Despite the apparent two-state equilibrium unfolding behavior during chemical denaturation, the kinetic unfolding pathway of MJ0366 involves the dissociation of the homodimeric native state into a native-like monomeric intermediate followed by unfolding into a denatured state. Our results provide comprehensive structural information regarding the folding dynamics and kinetic pathways of MJ0366, whose small size is ideal for converging experimental and theoretical findings to better understand the underlying principles of the folding of knotted proteins. [ABSTRACT FROM AUTHOR]

Prior studies have indicated that MJ1099 from Methanocaldococcus jannaschii has roles in the biosynthesis of tetrahydromethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 Å resolution using anomalous scattering methods. The results indicate that MJ1099 is a member of the TIM-barrel superfamily and that it is a homohexamer. Bioinformatic analyses identified a potential active site that is highly conserved among MJ1099 homologs and the key amino acids involved were identified. The results presented here should guide further studies of MJ1099 including mechanistic studies and possibly the development of inhibitors that target the methanogenic archaea in the digestive tracts of humans and that are a source of the greenhouse gas methane. [ABSTRACT FROM AUTHOR]

Methane is a potent greenhouse gas that is generated and consumed in anaerobic environments through the energy metabolism of methanogens and anaerobic methanotrophic archaea (ANME), respectively. Coenzyme F430 is essential for methanogenesis, and a structural variant of F430, 172-methylthio-F430 (F430-2), is found in ANME and is presumably essential for the anaerobic oxidation of methane. Here we use liquid chromatography-high-resolution mass spectrometry to identify several new structural variants of F430 in the cell extracts of selected methanogens and ANME. Methanocaldococcus jannaschii and Methanococcus maripaludis contain an F430 variant (denoted F430-3) that has an M+ of 1,009.2781. This mass increase of 103.9913 over that of F430 corresponds to C3H4O2S and is consistent with the addition of a 3-mercaptopropionate moiety bound as a thioether followed by a cyclization. The UV absorbance spectrum of F430-3 was different from that of F430 and instead matched that of an F430 derivative where the 17³ keto moiety had been reduced. This is the first report of a modified F430 in methanogens. In a search for F430-2 and F430-3 in other methanogens and ANME, we have identified a total of nine modified F430 structures. One of these compounds may be an abiotic oxidative product of F430, but the others represent naturally modified versions of F430. This work indicates that F430-related molecules have additional functions in nature and will inspire further research to determine the biochemical role(s) of these variants and the pathways involved in their biosynthesis. [ABSTRACT FROM AUTHOR]

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX3CX2C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor. [ABSTRACT FROM AUTHOR]

The gene encoding 7,8-dihydroneopterin aldolase (DHNA) was recently identified in archaea through comparative genomics as being involved in methanopterin biosynthesis (V. Crécy-Lagard, G. Phillips, L. L. Grochowski, B. El Yacoubi, F. Jenney, M. W. Adams, A. G. Murzin, and R. H. White, ACS Chem. Biol. 7:1807-1816, 2012, doi:10.1021/cb300342u). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a kcat/Km above 105 M-1 s-1 at 70°C, and at room temperature it exhibits a turnover number (0.07 s -1) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in kcat. An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gin caused a ≥25-fold increase in Km. These results provide important insights into the catalytic mechanism of archaeal DHNAs. [ABSTRACT FROM AUTHOR]

One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the β-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for β-alanine biosynthesis, we have established the pathway for the formation of β-alanine in this organism after experimentally eliminating other known and proposed pathways to β-alanine from malonate semialdehyde, L-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of β-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing β-alanine from L-aspartate is a pyruvoyl-containing L-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent L-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A Km of ~0.80 mM for L-aspartate with a specific activity of 0.09 µmol min-1 mg-1 at 70°C for the decarboxylation of L-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia colipanD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo. [ABSTRACT FROM AUTHOR]

Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme a-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of D2Okcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS (D2Okcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes. [ABSTRACT FROM AUTHOR]

We have established the biosynthetic pathway and the associated genes for the biosynthesis of the 5-(aminomethyl)-3-furanmethanol (F1) moiety of methanofuran in the methanogenic archaeon Methanocaldococcus jannaschii. The recombinant enzyme, derived from the MJ1099 gene, was shown to readily condense glyceraldehyde 3-phosphate (Ga-3P) and dihydroxyacetone-P (DHAP) to form 4-(hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P). The recombinant purified pyridoxal 5'-phosphate-dependent aminotransferase, derived from the MJ0684 gene, was found to be specific for catalyzing the transamination reaction between 4-HFC-P and [15N]alanine to produce [15N] 5-(aminomethyl)-3-furanmethanol-P (F1-P) and pyruvate. To confirm these results in cell extracts, we developed sensitive analytical methods for the liquid chromatography-ultraviolet-electrospray ionization mass spectrometry analysis of F1 as a 7-nitrobenzofurazan derivative. This method has allowed for the quantitation of trace amounts of F1 and F1-P in cell extracts and the measurement of the incorporation of stable isotopically labeled precursors into F1. After incubation of cell extracts with [1,2,3-13C3]pyruvate and DHAP, 4-([2H2]hydroxymethyl)-2-furancarboxylic acid phosphate (4-HFCA-P) or 4-([2H2]hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P) was found to be incorporated into F1-P. 4-HFCA-P and 4-HFC-P were confirmed in cell extracts after removal of the phosphate. The low level of incorporation of [1,2,3-13C3]pyruvate into F1-P in these experiments is explained by the fact that the labeled pyruvate must first be converted into Ga-3-P through gluconeogenesis before being incorporated into 4-HFC-P. Cell extracts incubated with 4-HFC-P and a mixture of [15N]aspartate, [15N]glutamate, and [15N]alanine produced [15N]F1-P. We also demonstrated that aqueous solutions of methylglyoxal or pyruvate heated with dihydroxyacetone led to the formation of 4-HFC and 4-HFCA, suggesting a possible prebiotic route to this moiety of methanofuran. [ABSTRACT FROM AUTHOR]

The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit-subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV. [ABSTRACT FROM AUTHOR]

Na+/H+ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na+/H+ exchangers, where a single binding site is alternatively occupied by Na+ or one or two H+ ions. The proposed transport mechanism inherently down-regulates Na+/H+ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na+/H+ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na+/H+ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na+/H+ exchanger from Escherichia coli (EcN-haA, shown in a previous publication) we conclude that it applies generally to all Na+/H+ exchangers, electrogenic as well as electro-neutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na+/H+ exchangers. [ABSTRACT FROM AUTHOR]

The crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii with the deletion of a nonconserved positively charged cluster consisting of eight C-terminal amino acid residues is determined by the molecular replacement method at 1.75 Å resolution. This mutant is shown to form more stable and ordered crystals belonging to a space group other than that of the wild-type protein crystals. The positively charged C-terminal region has only a slight effect on the interaction between protein L1 and RNA molecules. Hence, this mutant can be used to prepare protein-RNA complexes and obtain their crystals. [ABSTRACT FROM AUTHOR]

The aconitase family of proteins includes three classes of hydro-lyase enzymes: aconitases, homoaconitases and isopropylmalate (IPM) isomerases. They have a common Fe-S cluster-binding site and catalyze the isomerization of specific substrates by sequential dehydration and hydration. The archaeon Methanococcus jannaschii contains two aconitase family proteins, IPM isomerase and homoaconitase, which have 50% sequence identity. These two enzymes are heterodimeric proteins composed of large and small subunits encoded by separate genes. Although structures have been reported for the small subunits of the two enzymes, the first structures of oxidized and reduced forms of the large subunit of IPM isomerase (ox-MJ0499 and red-MJ0499, respectively) from M. jannaschii are reported here at 1.8 and 2.7 Å resolution, respectively, together with the structure of the large subunit of homoaconitase (MJ1003) at 2.5 Å resolution. The structures of both proteins have unbound Fe-S clusters and contain a fourth cysteine in the active site. The active site of MJ1003 is homologous to that of aconitase, whereas MJ0499 has significant structural distortion at the active site compared with aconitase. In addition, significant large conformational changes were observed in the active site of red-MJ0499 when compared with ox-MJ0499. The active sites of the two proteins adopt two different states before changing to the Fe-S cluster-bound 'activated' state observed in aconitase. MJ1003 has an 'open' active site, which forms an active pocket for the cluster, while ox-MJ0499 has a 'closed' active site, with four cysteines in disulfide bonds. These data will be helpful in understanding the biochemical mechanism of clustering of the Fe-S protein family. [ABSTRACT FROM AUTHOR]

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5'-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes conversion of 5'-deoxyadenosine to 5'-deoxyinosine as its major product but will also deaminate 5'-methylthioaden S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5'-deoxyadenosine deaminase (DadD). The Km for 5'-deoxyadenosine was found to be 14.0 ± 1.2 µM with a kcat/Km of 9.1 x 109 M-1 s-1. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5'-deoxyadenosine. Since 5'-dA has been demonstrated to be an inhibitor of radical SAM enzymes pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5'-deoxyadenosine, whereupon the 5'-deoxyribose moiety of 5'-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens. [ABSTRACT FROM AUTHOR]

Thermosipho globiformans (rod-shaped thermophilic fermenter) and Methanocaldococcus jannaschii (coccal hyperthermophilic hydrogenotrophic methanogen) established H-mediated syntrophy at 68 °C, forming exopolysaccharide-based aggregates. Electron microscopy showed that the syntrophic partners connected to each other directly or via intercellular bridges made from flagella, which facilitated transfer of H. Elemental sulfur (S) interrupted syntrophy; polysulfides abiotically formed from S intercepted electrons that were otherwise transferred to H to produce H, resulting in the generation of sulfide (sulfur respiration). However, Fe(III) oxides significantly reduced the interruption by S, accompanied by stiffening of Fe(II) sulfides produced by the reduction of Fe(III) oxides with the sulfur respiration-generated sulfide. Sea sand replacing Fe(III) oxides failed to generate stiffening or protect the syntrophy. Several experimental results indicated that the stiffening of Fe(II) sulfides shielded the liquid from S, resulting in methane production in the liquid. Field-emission scanning electron microscopy showed that the stiffened Fe(II) sulfides formed a network of spiny structures in which the microorganisms were buried. The individual fermenter rods likely produced Fe(II) sulfides on their surface and became local centers of a core of spiny structures, and the connection of these cores formed the network, which was macroscopically recognized as stiffening. [ABSTRACT FROM AUTHOR]