Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues*.

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.

The Oligosaccharyltransferase AglB Supports Surface-Associated Growth and Iron Oxidation in Methanococcus maripaludis.

Most microbial organisms grow as surface-attached communities known as biofilms. However, the mechanisms whereby methanogenic archaea grow attached to surfaces have remained understudied. Here, we show that the oligosaccharyltransferase AglB is essential for growth of Methanococcus maripaludis strain JJ on glass or metal surfaces. AglB glycosylates several cellular structures, such as pili, archaella, and the cell surface layer (S-layer). We show that the S-layer of strain JJ, but not strain S2, is a glycoprotein, that only strain JJ was capable of growth on surfaces, and that deletion of aglB blocked S-layer glycosylation and abolished surface-associated growth. A strain JJ mutant lacking structural components of the type IV-like pilus did not have a growth defect under any conditions tested, while a mutant lacking the preflagellin peptidase (ΔflaK) was defective for surface growth only when formate was provided as the sole electron donor. Finally, for strains that are capable of Fe0 oxidation, we show that deletion of aglB decreases the rate of anaerobic Fe0 oxidation, presumably due to decreased association of biomass with the Fe0 surface. Together, these data provide an initial characterization of surface-associated growth in a member of the methanogenic archaea. IMPORTANCE Methanogenic archaea are responsible for producing the majority of methane on Earth and catalyze the terminal reactions in the degradation of organic matter in anoxic environments. Methanogens often grow as biofilms associated with surfaces or partner organisms; however, the molecular details of surface-associated growth remain uncharacterized. We have found evidence that glycosylation of the cell surface layer is essential for growth of M. maripaludis on surfaces and can enhance rates of anaerobic iron corrosion. These results provide insight into the physiology of surface-associated methanogenic organisms and highlight the importance of surface association for anaerobic iron corrosion.

Effect of changes at the conserved + 3 position of mature archaellins on in vitro cleavage by the pre-archaellin peptidase FlaK of Methanococcus maripaludis.

Archaea swim using archaella that are domain-specific rotary type IV pilus-like appendages. The structural components of the archaellum filament are archaellins, initially made as preproteins with type IV pilin-like signal peptides which are removed by signal peptidases that are homologues of prepilin peptidases that remove signal peptides from type IV pilins. N-terminal sequences of archaellins, including the signal peptide cleavage site, are conserved and various positions have been previously shown to be critical for signal peptide removal. Archaellins have an absolute conservation of glycine at the + 3 position from the signal peptide cleavage site. To investigate its role in signal peptide cleavage, I used archaellin variants in which the + 3 glycine was mutated to all other possibilities in in vitro cleavage reactions. Cleavage was observed with ten different amino acids at the + 3 position, indicating that the observed glycine conservation is not required for this essential processing step.

A New Class of Phosphoribosyltransferases Involved in Cobamide Biosynthesis Is Found in Methanogenic Archaea and Cyanobacteria.

Cobamides are coenzymes used by cells from all domains of life but made de novo by only some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica ( SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii ( MjCobT) is known, but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTases. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.

Parts-Prospecting for a High-Efficiency Thiamin Thiazole Biosynthesis Pathway.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

Identification and characterization of the 4-epimerase AglW from the archaeon Methanococcus maripaludis.

Archaea are ubiquitous single-cell microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis, a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with N-linked tetrasaccharides that are essential for the proper assembly and function of archaella. The aglW gene, encoding the putative 4-epimerase AglW, plays a key role in the synthesis of the tetrasaccharide. The goal of our work was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop assays to determine its substrate specificity and properties. We carried out assays using UDP-Galactose, UDP-Glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and N-acetylglucosamine/N-acetylgalactosamine-diphosphate - lipid as substrates, coupled with specific glycosyltransferases. We showed that AglW has a broad specificity towards UDP-sugars and that Tyr151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The glycosyltransferase-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases.

Methanococcus maripaludis Employs Three Functional Heterodisulfide Reductase Complexes for Flavin-Based Electron Bifurcation Using Hydrogen and Formate.

Hydrogenotrophic methanogens oxidize molecular hydrogen to reduce carbon dioxide to methane. In methanogens without cytochromes, the initial endergonic reduction of CO2 to formylmethanofuran with H2-derived electrons is coupled to the exergonic reduction of a heterodisulfide of coenzymes B and M by flavin-based electron bifurcation (FBEB). In Methanococcus maripaludis, FBEB is performed by a heterodisulfide reductase (Hdr) enzyme complex that involves hydrogenase (Vhu), although formate dehydrogenase (Fdh) has been proposed as an alternative to Vhu. We have identified and purified three Hdr complexes of M. maripaludis, where homodimeric Hdr complexes containing (Vhu)2 or (Fdh)2 were found, in addition to a heterocomplex that contains both Vhu and Fdh. Formate was found in in vitro assays using the purified Hdr complex to act directly as the electron donor for FBEB via the associated Fdh. Furthermore, while ferredoxin was slowly reduced to 30% [-360 mV vs the standard hydrogen electrode (SHE)] by H2 and formate (0.8 atm and 30 mM, according to thermodynamics), the addition of CoB-S-S-CoM as the high-potential electron acceptor ( E°' = -140 mV vs SHE; to induce FBEB) resulted in the rapid and more complete reduction of Fd to 94% (-455 mV vs SHE).

Genetically Encoded Protein Phosphorylation in Mammalian Cells.

Protein phosphorylation regulates diverse processes in eukaryotic cells. Strategies for installing site-specific phosphorylation in target proteins in eukaryotic cells, through routes that are orthogonal to enzymatic post-translational modification, would provide a powerful route for defining the consequences of particular phosphorylations. Here we show that the SepRSv1.0/tRNAv1.0CUA pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synthetase [MmSepRS]/Methanococcus janaschii [Mj]tRNAGCACys pair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1α) variant, EF-1α-Sep, that enhances phosphoserine incorporation, and combine this with a mutant of eRF1, and manipulations of the cell's phosphoserine biosynthetic pathway, to enable the genetically encoded incorporation of phosphoserine and its non-hydrolyzable phosphonate analog. Using this approach we demonstrate synthetic activation of a protein kinase in mammalian cells.

Assembly of Methyl Coenzyme M Reductase in the Methanogenic Archaeon Methanococcus maripaludis.

Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile Methanothermococcus okinawensis (rMCRok) was expressed in the mesophile Methanococcus maripaludis The rMCRok was posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F430 Subunits of the native M. maripaludis (MCRmar) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-tagged mcrA from M. maripaludis and the mcrBDCG from M. okinawensis in M. maripaludis The His-tagged purified rMCR then contained the M. maripaludis McrA and the M. okinawensis McrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.IMPORTANCE Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts for about 74% of the total global methane production, up to 25% of which is consumed by anaerobic oxidation of methane (AOM). Methyl coenzyme M reductase (MCR) is the key enzyme in both methanogenesis and AOM. MCR is assembled as a dimer of two heterotrimers, where posttranslational modifications and F430 cofactors are embedded in the active sites. However, this complex assembly process remains unknown. Here, we established a heterologous expression system for MCR to learn how MCR is assembled.

Identification and Biosynthesis of 1-Mercaptoethanesulfonic Acid (1-MES), an Analogue of Coenzyme M, Found Widely in the Methanogenic Archaea.

Here I report on the identification of 1-mercaptoethanesulfonic acid (1-MES), an analogue of 2-mercaptoethanesulfonic acid (coenzyme M, HSCoM). 1-MES and HSCoM were both present in the growth media of eight different methanogens at concentrations ranging from ∼1 to 100 µM. In an effort to determine a chemical origin of 1-MES, several plausible chemical routes were examined each assuming that HSCoM was the precursor. In all examined routes, no 1-MES was formed. However, 1-MES was formed when a solution of vinylsulfonic acid and sulfide were exposed to ultraviolet light. On the basis of these results, I conclude 1-MES is formed enzymatically. This was confirmed by growing a culture of Methanococcus maripaludis S2 in the presence of [1,1',2,2'-2H4]HSCoM and measuring the incorporation of deuterium into 1-MES. 1-MES incorporated three of the four deuteriums from the fed HSCoM. This result is consistent with the abstraction of a C-2 deuterium of the HSCoM, likely by a 5'-dAdoCH2• radical, followed by a radical rearrangement in which the sulfonic acid moves to position C-1, followed by abstraction of a H• likely from 5'-dAdoCH2D. At present, the reason for the production of 1-MES is not clear. This is the first report of the occurrence of 1-MES in Nature.

Structural Basis for the Persistence of Homing Endonucleases in Transcription Factor IIB Inteins.

Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.

The crystal structure of the Pyrococcus abyssi mono-functional methyltransferase PaTrm5b.

The wyosine hypermodification found exclusively at G37 of tRNAPhe in eukaryotes and archaea is a very complicated process involving multiple steps and enzymes, and the derivatives are essential for the maintenance of the reading frame during translation. In the archaea Pyrococcus abyssi, two key enzymes from the Trm5 family, named PaTrm5a and PaTrm5b respectively, start the process by forming N1-methylated guanosine (m1G37). In addition, PaTrm5a catalyzes the further methylation of C7 on 4-demethylwyosine (imG-14) to produce isowyosine (imG2) at the same position. The structural basis of the distinct methylation capacities and possible conformational changes during catalysis displayed by the Trm5 enzymes are poorly studied. Here we report the 3.3 Å crystal structure of the mono-functional PaTrm5b, which shares 32% sequence identity with PaTrm5a. Interestingly, structural superposition reveals that the PaTrm5b protein exhibits an extended conformation similar to that of tRNA-bound Trm5b from Methanococcus jannaschii (MjTrm5b), but quite different from the open conformation of apo-PaTrm5a or well folded apo-MjTrm5b reported previously. Truncation of the N-terminal D1 domain leads to reduced tRNA binding as well as the methyltransfer activity of PaTrm5b. The differential positioning of the D1 domains from three reported Trm5 structures were rationalized, which could be attributable to the dissimilar inter-domain interactions and crystal packing patterns. This study expands our understanding on the methylation mechanism of the Trm5 enzymes and wyosine hypermodification.

A novel double kink-turn module in euryarchaeal RNase P RNAs.

RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented 'double K-turn' module in type A and type M archaeal RPR variants.

Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases.

The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.

Identification of inhibitors of the transmembrane protease FlaK of Methanococcus maripaludis.

GxGD-type intramembrane cleaving proteases (I-CLiPs) form a family of proteolytic enzymes that feature an aspartate-based catalytic mechanism. Yet, they structurally and functionally largely differ from the classical pepsin-like aspartic proteases. Among them are the archaeal enzyme FlaK, processing its substrate FlaB2 during the formation of flagella and γ-secretase, which is centrally involved in the etiology of the neurodegenerative Alzheimer's disease. We developed an optimized activity assay for FlaK and based on screening of a small in-house library and chemical synthesis, we identified compound 9 as the first inhibitor of this enzyme. Our results show that this intramembrane protease differs from classical pepsin-like aspartic proteases and give insights into the substrate recognition of this enzyme. By providing the needed tools to further study the enzymatic cycle of FlaK, our results also enable further studies towards a functional understanding of other GxGD-type I-CLiPs.

Identification of a gene involved in the biosynthesis pathway of the terminal sugar of the archaellin N-linked tetrasaccharide in Methanococcus maripaludis.

In Methanococcus maripaludis, the three archaellins which comprise the archaellum are modified at multiple sites with an N-linked tetrasaccharide with the structure of Sug-4-ß-ManNAc3NAmA6Thr-4-ß-GlcNAc3NAcA-3-ß-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose, so far found exclusively in this species. In this study, a six-gene cluster mmp1089-1094, neighboring one of the genomic regions already known to contain genes involved with the archaellin N-glycosylation pathway, was examined for its potential involvement in the archaellin N-glycosylation or sugar biosynthesis pathway. The co-transcription of these six genes was demonstrated by RT-PCR. Mutants carrying an in-frame deletion in mmp1090, mmp1091 or mmp1092 were successfully generated. The Δmmp1090 deletion mutant was archaellated when examined by electron microscopy and mass spectrometry analysis of purified archaella showed that the archaellins were modified with a truncated N-glycan in which the terminal sugar residue and the threonine linked to the third sugar residue were missing. Both gene annotation and bioinformatic analyses indicate that MMP1090 is a UDP-glucose 4-epimerase, suggesting that the unique terminal sugar of the archaellin N-glycan might be synthesised from UDP-glucose or UDP-N-acetylglucosamine with an essential early step in synthesis catalysed by MMP1090. In contrast, no detectable phenotype related to archaellin glycosylation was observed in mutants deleted for either mmp1091 or mmp1092 while attempts to delete mmp1089, mmp1093 and mmp1094 were unsuccessful. Based on its demonstrated involvement in the archaellin N-glycosylation pathway, we designated mmp1090 as aglW.

Alpha-ring Independent Assembly of the 20S Proteasome.

Archaeal proteasomes share many features with their eukaryotic counterparts and serve as important models for assembly. Proteasomes are also found in certain bacterial lineages yet their assembly mechanism is thought to be fundamentally different. Here we investigate α-ring formation using recombinant proteasomes from the archaeon Methanococcus maripaludis. Through an engineered disulfide cross-linking strategy, we demonstrate that double α-rings are structurally analogous to half-proteasomes and can form independently of single α-rings. More importantly, via targeted mutagenesis, we show that single α-rings are not required for the efficient assembly of 20S proteasomes. Our data support updating the currently held "α-ring first" view of assembly, initially proposed in studies of archaeal proteasomes, and present a way to reconcile the seemingly separate bacterial assembly mechanism with the rest of the proteasome realm. We suggest that a common assembly network underpins the absolutely conserved architecture of proteasomes across all domains of life.

Study of the Binding Energies between Unnatural Amino Acids and Engineered Orthogonal Tyrosyl-tRNA Synthetases.

We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs.

Mechanistic Studies of the Radical S-Adenosyl-L-methionine Enzyme 4-Demethylwyosine Synthase Reveal the Site of Hydrogen Atom Abstraction.

TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m¹G) with carbons 2 and 3 of pyruvate. In this study, labeled transfer ribonucleic acid (tRNA) and pyruvate were utilized to determine the site of hydrogen atom abstraction and regiochemistry of the pyruvate addition. tRNA containing a ²H-labeled m¹G methyl group was used to identify the methyl group of m¹G as the site of hydrogen atom abstraction by 5'-deoxyadenosyl radical. [2-¹³C1-3,3,3-²H3]Pyruvate was used to demonstrate retention of all the pyruvate protons, indicating that C2 of pyruvate forms the bridging carbon of the imidazoline ring and C3 the methyl.

Intrinsic disorder and metal binding in UreG proteins from Archae hyperthermophiles: GTPase enzymes involved in the activation of Ni(II) dependent urease.

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii (Mj) and Metallosphaera sedula (Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG.