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tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5ʹ-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5ʹ-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 A resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5ʹ-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π∗ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

Formation of the internal aldimine (LLP) is the first regulatory step that activates pyridoxal 5′-phosphate (PLP) dependent enzymes. The process involves a nucleophilic attack on PLP by an active site Lys residue, followed by proton transfers resulting in a carbinolamine (CBA) intermediate that undergoes dehydration to form the aldimine. Despite a general understanding of the pathway, the structural basis of the mechanistic roles of specific residues in each of these steps is unclear. Here we determined the crystal structure of the LLP form (holo-form) of a Group II PLP-dependent decarboxylase from Methanocaldococcus jannaschii (MjDC) at 1.7 A resolution. By comparing the crystal structure of MjDC in the LLP form with that of the pyridoxal-P (non-covalently bound aldehyde) form, we demonstrate structural evidence for a water-mediated mechanism of LLP formation. A conserved extended hydrogen-bonding network around PLP coupled to the pyridinyl nitrogen influences activation and catalysis by affecting the electronic configuration of PLP. Furthermore, the two cofactor bound forms revealed open and closed conformations of the catalytic loop (CL) in the absence of a ligand, supporting a hypothesis for a regulatory link between LLP formation and CL dynamics. The evidence suggests that activation of Group II decarboxylases involves a complex interplay of interactions between the electronic states of PLP, the active site micro-environment and CL dynamics.

Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time.

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., Graphical abstract, Highlights • CorA channels regulate magnesium ion concentration in the bacterial cytoplasm. • We report the first structural data on CorA when depleted of magnesium ions. • The absence of magnesium ions leads to conformational changes in the channel. • CorA structure is influenced by ionic bonds between channel domains. • CorA structure is influenced by monovalent ions binding to cytoplasmic domains.

Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated kat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4 x 10(-5) s(-1). Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450 +/- 20 mu g/ml CAT109pAzF and 428 +/- 2 mu g/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe. (C) 2012 Elsevier Inc. All rights reserved.

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric alpha-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the alpha-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the alpha-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an "all-or-none" mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the alpha-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the alpha-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.

In mammals, 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (alpha) and two regulatory subunits (beta and gamma). The gamma-subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. We describe herein the crystal structure of protein MJ1225 from Methanocaldococcus jannaschii complexed to AMP, ADP, and ATP. Our data provide evidence of a strong conservation of the key functional features seen in the gamma-subunit of the eukaryotic AMPK, and more importantly, it reveals a novel AMP binding site, herein denoted as site E, which had not been previously described in cystathionine beta-synthase domains so far. Site E is located in a small cavity existing between the alpha-helices structurally equivalent to those disrupting the internal symmetry of each Bateman domain in gamma-AMPKs and shows striking similarities with a symmetry-related crevice of the mammalian enzyme that hosts the pathological mutation N488I.

We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.

Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as (S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6 M guanidine-HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110 kDa. NADP was preferred as a coenzyme over NAD for (S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2S,3S)-tartrate and (2S,3R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archaea Methanocaldococcus jannaschii and Archaeoglobus fulgidus, suggesting these hyperthermophilic MDHs loosely bind their substrates. The refolded A. pernix MDH was also crystallized, and the structure was determined at a resolution of 2.9 A. Its overall structure was similar to those of the M. jannaschii, Chloroflexus aurantiacus, Chlorobium vibrioforme and Cryptosporidium parvum [lactate dehydrogenase-like] MDHs with root-mean-square-deviation values between 1.4 and 2.1 A. Consistent with earlier reports, Ala at position 53 was responsible for coenzyme specificity, and the next residue, Arg, was important for NADP binding. Structural comparison revealed that the hyperthermostability of the A. pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme.

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported. Here, we report the structure of Dim1 from the thermophilic archaeon Methanocaldococcus jannaschii. While it shares obvious similarities with the bacterial and eukaryotic orthologs, notable structural differences exist among the three members, particularly in the C-terminal domain. Previous work showed that eukaryotic and archaeal Dim1 were able to robustly complement for KsgA in E. coli. Here, we repeated similar experiments to test for complementarity of archaeal Dim1 and bacterial KsgA in Saccharomyces cerevisiae. However, neither the bacterial nor the archaeal ortholog could complement for the eukaryotic Dim1. This might be related to the secondary, non-methyltransferase function that Dim1 is known to play in eukaryotic ribosomal maturation. To further delineate regions of the eukaryotic Dim1 critical to its function, we created and tested KsgA/Dim1 chimeras. Of the chimeras, only one constructed with the N-terminal domain from eukaryotic Dim1 and the C-terminal domain from archaeal Dim1 was able to complement, suggesting that eukaryotic-specific Dim1 function resides in the N-terminal domain also, where few structural differences are observed between members of the KsgA/Dim1 family. Future work is required to identify those determinants directly responsible for Dim1 function in ribosome biogenesis. Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures.

In the archaebacterium Methanocaldococcus jannaschii (M. jannaschii), the proteasomal regulatory particle (RP), a homohexameric complex of proteasome-activating nucleotidase (PAN), is responsible for target protein recognition, followed by unfolding and translocation of the bound protein into the core particle (CP) for degradation. Guided by structure-based mutagenesis, we identify amino acids and structural motifs that are essential for PAN function. Key residues line the axial channel of PAN, defining the apparent pathway of substrate translocation. Subcomplex II of PAN, comprising the ATPase domain, associates with the CP and drives ATP-dependent unfolding of the substrate protein, whereas the distal subcomplex I forms the entry port of the substrate translocation channel. A linker segment between subcomplexes I and II is essential for PAN function, implying functional and perhaps mechanical coupling between these domains. Sequence conservation suggests that the principles of PAN function are likely to apply to the proteasomal RP of eukaryotes.

Carbonaceous matter (CM) from ca. 3.5 Ga hydrothermal black cherts of the Pilbara Craton of Western Australia and the Barberton Greenstone Belt of South Africa yielded transmission electron microscopy (TEM) images that are suggestive of microbial remains and possible remnants of microbial cell walls. These are compared to a potential modern analogue, the hyperthermophilic Methanocaldococcus jannaschii, derived from an active seafloor hydrothermal environment and cultured under similar conditions. A striking resemblance to the early Archaean forms was evident in wall structure and thermal degradation mode. Cell disintegration of the cultures occurred at 100 °C marking the limits of life. Complete disintegration, deformation and shrinkage occurred at 132 °C. A multidisciplinary approach to the characterisation of the CM was undertaken using organic petrology, TEM coupled with electron dispersive spectral analysis (EDS), high resolution TEM (HRTEM) to determine molecular ordering, and elemental and carbon isotope geochemistry. Reflectance measurements of the CM to determine thermal stress yielded a range of values corresponding to several populations, and pointing to different sources and processes. The δ13C values of Dresser Formation CM (−36.5 to −32.1‰) are negatively correlated with TOC (0.13–0.75%) and positively correlated with C/N ratio (134–569), which is interpreted to reflect the relative abundance of high Ro/oxidised/recycled CM and preferential loss of 12C and N during thermal maturation. TEM observations, inferred carbon isotopic heterogeneity and isotope fractionations of −27 to −32‰ are consistent with the activity of chemosynthetic microbes in a seafloor hydrothermal system where rapid silicification at relatively low temperature preserved the CM.

Chaperone function in water-miscible organic co-solvents is useful for biocatalytic applications requiring enzyme stability in semi-aqueous media and for understanding chaperone behavior in hydrophobic environments. Previously, we have shown that a recombinant single subunit thermosome (rTHS) from Methanocaldococcus jannaschii functions in multiple co-solvents to hydrolyze ATP, prevent protein aggregation, and refold enzymes following solvent denaturation. For the present study, a truncated analog to the thermosome in which 70 N-terminal amino acids are removed is used to identify important regions within the thermosome for its chaperoning functions in organic co-solvents. Data presented herein indicate that the N-terminal region of rTHS is essential for the chaperone to restore the native state of the enzyme citrate synthase, but it is not a critical region for either binding of unfolded proteins or ATP hydrolysis. This is the first demonstration that direct refolding by a Group II chaperonin requires the N-terminal region of the protein.

Molecular chaperones play a central role in maintaining protein structure within a cell. Previously, we determined that the gene encoding a molecular chaperone, a thermosome, from the hyperthermophilic archaeon Methanocaldococcus jannaschii is upregulated upon lethal heat shock. We have recombinantly expressed this thermosome (rTHS) and show here that it is both stable and fully functional in aqueous solutions containing water-miscible organic co-solvents. Based on circular dichroism the secondary structure of rTHS was not affected by one-hour exposures to a variety of co-solvents including 30% v/v acetonitrile (ACN) and 50% methanol (MeOH). By contrast, the secondary structure of a mesophilic homologue, GroEL/GroES (GroE), was substantially disrupted. rTHS reduced the aggregation of ovalbumin and citrate synthase in 30% ACN, assisted refolding of citrate synthase upon solvent-inactivation, and stabilized citrate synthase and glutamate dehydrogenase in the direct presence of co-solvents. Apparent total turnover numbers of these enzymes in denaturing solutions increased by up to 2.5-fold in the presence of rTHS. Mechanistic models are proposed to help ascertain specific conditions that could enhance or limit organic solvent-induced chaperone activity. These models suggest that thermodynamic stability and the reversibility of enzyme unfolding play key roles in the effectiveness of enzyme recovery by rTHS.

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1–145) is similar to cytosine deaminase and the C-terminal domain (146–367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP+) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159–173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C′/D′ motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or “remodeling” as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7–sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6′s distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.

Coenzyme A as the principal acyl carrier is required for many synthetic and degradative reactions in intermediary metabolism. It is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes of eubacteria, plants, and human were all identified and cloned. In most bacteria, the so-called Dfp proteins catalyze the synthesis of the coenzyme A precursor 4'-phosphopantetheine. Dfp proteins are bifunctional enzymes catalyzing the synthesis of 4'-phosphopantothenoylcysteine (CoaB activity) and its decarboxylation to 4'-phosphopantetheine (CoaC activity). Here, we demonstrate the functional characterization of the CoaB and CoaC domains of an archaebacterial Dfp protein. Both domains of the Methanocaldococcus jannaschii Dfp protein were purified as His tag proteins, and their enzymatic activities were then identified and characterized by site-directed mutagenesis. Although the nucleotide binding motif II of the CoaB domain resembles that of eukaryotic enzymes, Methanocaldococcus CoaB is a CTP- and not an ATP-dependent enzyme, as shown by detection of the 4'-phosphopantothenoyl-CMP intermediate. The proposed 4'-phosphopantothenoylcysteine binding clamp of the Methanocaldococcus CoaC activity differs significantly from those of other characterized CoaC proteins. In particular, the active site cysteine residue, which otherwise is involved in the reduction of an aminoenethiol reaction intermediate, is not present. Moreover, the conserved Asn residue of the PXMNXXMW motif, which contacts the carboxyl group of 4'-phosphopantothenoylcysteine, is exchanged for His.

Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.

Methanofuran is the first coenzyme in the methanogenic pathway used by the archaeon Methanocaldococcus jannaschii , as well as other methanogens, to reduce CO 2 to methane. The details of the pathway for the biosynthesis of methanofuran and the responsible genes have yet to be established. A clear structural element in all known methanofurans is tyramine, likely produced by the decarboxylation of l -tyrosine. We show here that the mfnA gene at M. jannaschii locus MJ0050 encodes a thermostable pyridoxal phosphate-dependent l -tyrosine decarboxylase that specifically produces tyramine. Homologs of this gene are widely distributed among euryarchaea but are not specifically related to known bacterial or plant tyrosine decarboxylases.

We have identified and characterized a new member of the ATP-grasp enzyme family that catalyzes the ATP- and formate-dependent formylation of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (AICAR) to 5-formaminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (FAICAR) in the absence of folates. The enzyme, which we designate as PurP, is the product of the Methanocaldococcus jannaschii purP gene (MJ0136), which is a signature gene for Archaea. As is characteristic of reactions catalyzed by this family of enzymes, the other products of the reaction, ADP and Pi, were produced stoichiometrically with the amount of ATP, formate, and AICAR used. Formyl phosphate was found to substitute for ATP and formate in the reaction, yet the methylene analog, phosphonoacetaldehyde, was not an inhibitor or substrate for the reaction. The enzyme, along with PurO, which catalyzes the cyclization of FAICAR to inosine 5′-monophosphate, catalyzes the same overall transformation in purine biosynthesis as is accomplished by PurH in bacteria and eukaryotes. No homology exists between PurH and either PurO or PurP. 1H NMR and gas chromatography-mass spectrometry analysis of an M. jannaschii cell extract showed the presence of free formate that can be used by the enzyme for purine biosynthesis. This formate arises by the reduction of CO2 with hydrogen; this was demonstrated by incorporating 13C into the formate when M. jannaschii cell extracts were incubated with and hydrogen gas. The presence of this signature gene in all of the Archaea indicates the presence of a purine biosynthetic pathway proceeding in the absence of folate coenzymes.

Borrelidin, a compound with anti-microbial and anti-angiogenic properties, is a known inhibitor of bacterial and eukaryal threonyl-tRNA synthetase (ThrRS). The inhibition mechanism of borrelidin is not well understood. Archaea contain archaeal and bacterial genre ThrRS enzymes that can be distinguished by their sequence. We explored species-specific borrelidin inhibition of ThrRSs. The activity of ThrRS from Sulfolobus solfataricus and Halobacterium sp. NRC-1 was inhibited by borrelidin, whereas ThrRS enzymes from Methanocaldococcus jannaschii and Archaeoglobus fulgidus were not. In Escherichia coli ThrRS, borrelidin binding induced a conformational change, and threonine was not activated as shown by ATP-PPi exchange and a transient kinetic assay measuring intrinsic tryptophan fluorescence changes. These assays further showed that borrelidin is a noncompetitive tight binding inhibitor of E. coli ThrRS with respect to threonine and ATP. Genetic selection of borrelidin-resistant mutants showed that borrelidin binds to a hydrophobic region (Thr-307, His-309, Cys-334, Pro-335, Leu-489, Leu-493) proximal to the zinc ion at the active site of the E. coli ThrRS. Mutating residue Leu-489 → Trp reduced the space of the hydrophobic cluster and resulted in a 1500-fold increase of the Ki value from 4 nm to 6 μm. An alignment of ThrRS sequences showed that this cluster is conserved in most organisms except for some Archaea (e.g. M. jannaschii, A. fulgidus) and some pathogens (e.g. Helicobacter pylori). This study illustrates how one class of natural product inhibitors affects aminoacyl-tRNA synthetase function, providing potentially useful information for structure-based inhibitor design.

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.

By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km100 micro m). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.

In this issue of Structure, Borowska et al. (2015) report the crystal structure and provide experimental evidence on an archaeal membrane insertase, the DUF106 protein from Methanocaldococcus jannaschii, demonstrating that YidC/Oxa1/Alb3-like insertases exist in the archaeal plasma membrane.

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from the in vitro assembly of the 12-subunit RNA polymerase from the hyperthermophile Methanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.