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PubMed1995

Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter.

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.

Base Sequence↗
PubMed1998

Structure-based assignment of the biochemical function of a hypothetical protein: a test case of structural genomics.

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms' predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and/or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile, Methanococcus jannaschii, at 1.7-A resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.

Adenosine Triphosphatases↗
PubMed1999

Domain-domain communication in a miniature archaebacterial tRNA synthetase.

The three-dimensional structure of tRNA is organized into two domains-the acceptor-TPsiC minihelix with the amino acid attachment site and a second, anticodon-containing, stem-loop domain. Aminoacyl-tRNA synthetases have a structural organization that roughly recapitulates the two-domain organization of tRNAs-an older primary domain that contains the catalytic center and interacts with the minihelix and a secondary, more recent, domain that makes contacts with the anticodon-containing arm. The latter contacts typically are essential for enhancement of the catalytic constant k(cat) through domain-domain communication. Methanococcus jannaschii tyrosyl-tRNA synthetase is a miniature synthetase with a tiny secondary domain suggestive of an early synthetase evolving from a one-domain to a two-domain structure. Here we demonstrate functional interactions with the anticodon-containing arm of tRNA that involve the miniaturized secondary domain. These interactions appear not to include direct contacts with the anticodon triplet but nonetheless lead to domain-domain communication. Thus, interdomain communication may have been established early in the evolution from one-domain to two-domain structures.

Acylation↗
PubMed1999

Detection of protein fold similarity based on correlation of amino acid properties.

An increasing number of proteins with weak sequence similarity have been found to assume similar three-dimensional fold and often have similar or related biochemical or biophysical functions. We propose a method for detecting the fold similarity between two proteins with low sequence similarity based on their amino acid properties alone. The method, the proximity correlation matrix (PCM) method, is built on the observation that the physical properties of neighboring amino acid residues in sequence at structurally equivalent positions of two proteins of similar fold are often correlated even when amino acid sequences are different. The hydrophobicity is shown to be the most strongly correlated property for all protein fold classes. The PCM method was tested on 420 proteins belonging to 64 different known folds, each having at least three proteins with little sequence similarity. The method was able to detect fold similarities for 40% of the 420 sequences. Compared with sequence comparison and several fold-recognition methods, the method demonstrates good performance in detecting fold similarities among the proteins with low sequence identity. Applied to the complete genome of Methanococcus jannaschii, the method recognized the folds for 22 hypothetical proteins.

Amino Acid Sequence↗
PubMed1998

Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.

Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.

Acids↗
PubMed1999

Studies on the ADP-ribose pyrophosphatase subfamily of the nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance.

Four Nudix hydrolase genes, ysa1 from Saccharomyces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite.

Adenosine Triphosphatases↗
PubMed1999

Major anticodon-binding region missing from an archaebacterial tRNA synthetase.

The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor.TpsiC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodon-binding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.

Anticodon↗
PubMed2000

Identification of a highly diverged class of S-adenosylmethionine synthetases in the archaea.

S-adenosylmethionine is the primary alkylating agent in all known organisms. ATP:L-methionine S-adenosyltransferase (MAT) catalyzes the only known biosynthetic route to this central metabolite. Although the amino acid sequence of MAT is strongly conserved among bacteria and eukarya, no homologs have been recognized in the completed genome sequences of any archaea. In this study, MAT has been purified to homogeneity from the archaeon Methanococcus jannaschii, and the gene encoding it has been identified by mass spectrometry. The peptide mass map identifies the gene encoding MAT as MJ1208, a hypothetical open reading frame. The gene was cloned in Escherichia coli, and expressed enzyme has been purified and characterized. This protein has only 22 and 23% sequence identity to the E. coli and human enzymes, respectively, whereas those are 59% identical to each other. The few identical residues include the majority of those constituting the polar active site residues. Each complete archaeal genome sequence contains a homolog of this archaeal-type MAT. Surprisingly, three bacterial genomes encode both the archaeal and eukaryal/bacterial types of MAT. This identification of a second major class of MAT emphasizes the long evolutionary history of the archaeal lineage and the structural diversity found even in crucial metabolic enzymes.

Amino Acid Sequence↗
PubMed2002

Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters.

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5'-(beta,gamma-imino)triphosphate and adenosine 5'-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction.

Adenosine Diphosphate↗
PubMed2000

Protein splicing in the absence of an intein penultimate histidine.

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.

Asparagine↗
PubMed2001

Activation of cell division protein FtsZ. Control of switch loop T3 conformation by the nucleotide gamma-phosphate.

The effect of bound nucleotide on the conformation of cell division protein FtsZ from Methanococcus jannaschii has been investigated using molecular dynamics and site-directed mutagenesis. The molecular dynamics indicate that the gamma-phosphate of GTP induces a conformational perturbation in loop T3 (Gly88-Gly99 segment), in a position structurally equivalent to switch II of Ha-ras-p21. In the simulated GTP-bound state, loop T3 is pulled by the gamma-phosphate into a more compact conformation than with GDP, related to that observed in the homologous proteins alpha- and beta-tubulin. The existence of a nucleotide-induced structural change in loop T3 has been confirmed by mutating Thr92 into Trp (T92W-W319Y FtsZ). This tryptophan (12 A away from gamma-phosphate) shows large differences in fluorescence emission, depending on which nucleotide is bound to FtsZ monomers. Loop T3 is located at a side of the contact interface between two FtsZ monomers in the current model of FtsZ filament. Such a structural change may bend the GDP filament upon hydrolysis by pushing against helix H8 of next monomer, thus, generating force on the membrane during cell division. A related curvature mechanism may operate in tubulin activation.

Amino Acid Sequence↗
PubMed2001

Species-specific differences in amino acid editing by class II prolyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates. Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups. We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well. Previously, we showed that some features of tRNA recognition and aminoacylation are indeed group-specific. Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing. Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins. "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid. We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity. Whereas ProRS from Methanococcus jannaschii is similar to E. coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities. These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs.

Alanine↗
PubMed2002

Identification of coenzyme M biosynthetic phosphosulfolactate synthase: a new family of sulfonate-biosynthesizing enzymes.

The hyperthermophilic euryarchaeon Methanococcus jannaschii uses coenzyme M (2-mercaptoethanesulfonic acid) as the terminal methyl carrier in methanogenesis. We describe an enzyme from that organism, (2R)-phospho-3-sulfolactate synthase (ComA), that catalyzes the first step in coenzyme M biosynthesis. ComA catalyzed the stereospecific Michael addition of sulfite to phosphoenolpyruvate over a broad range of temperature and pH conditions. Substrate and product analogs moderately inhibited activity. This enzyme has no significant sequence similarity to previously characterized enzymes; however, its Mg(2+)-dependent enzyme reaction mechanism may be analogous to one proposed for enolase. A diverse group of microbes and plants have homologs of ComA that could have been recruited for sulfolactate or sulfolipid biosyntheses.

Amino Acid Sequence↗
PubMed2002

Crystal structure of a dual activity IMPase/FBPase (AF2372) from Archaeoglobus fulgidus. The story of a mobile loop.

Several hyperthermophilic organisms contain an unusual phosphatase that has dual activity toward inositol monophosphates and fructose 1,6-bisphosphate. The structure of the second member of this family, an FBPase/IMPase from Archaeoglobus fulgidus (AF2372), has been solved. This enzyme shares many kinetic and structural similarities with that of a previously solved enzyme from Methanococcus jannaschii (MJ0109). It also shows some kinetic differences in divalent metal ion binding as well as structural variations at the dimer interface that correlate with decreased thermal stability. The availability of different crystal forms allowed us to investigate the effect of the presence of ligands on the conformation of a mobile catalytic loop independently of the crystal packing. This conformational variability in AF2372 is compared with that observed in other members of this structural family that are sensitive or insensitive to submillimolar concentrations of Li(+). This analysis provides support for the previously proposed mechanism of catalysis involving three metal ions. A direct correlation of the loop conformation with strength of Li(+) inhibition provides a useful system of classification for this extended family of enzymes.

5'-Nucleotidase↗
PubMed2002

Reversible unfolding of FtsZ cell division proteins from archaea and bacteria. Comparison with eukaryotic tubulin folding and assembly.

The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers.

Amino Acid Sequence↗
PubMed2003

Assembly of archaeal cell division protein FtsZ and a GTPase-inactive mutant into double-stranded filaments.

We have studied the assembly and GTPase of purified FtsZ from the hyperthermophilic archaeon Methanococcus jannaschii, a structural homolog of eukaryotic tubulin, employing wild-type FtsZ, FtsZ-His6 (histidine-tagged FtsZ), and the new mutants FtsZ-W319Y and FtsZ-W319Y-His6, with light scattering, nucleotide analyses, electron microscopy, and image processing methods. This has revealed novel properties of FtsZ. The GTPase of archaeal FtsZ polymers is suppressed in Na+-containing buffer, generating stabilized structures that require GDP addition for disassembly. FtsZ assembly is polymorphic. Archaeal FtsZ(wt) assembles into associated and isolated filaments made of two parallel protofilaments with a 43 A longitudinal spacing between monomers, and this structure is also observed in bacterial FtsZ from Escherichia coli. The His6 extension facilitates the artificial formation of helical tubes and sheets. FtsZ-W319Y-His6 is an inactivated GTPase whose assembly remains regulated by GTP and Mg2+. It forms two-dimensional crystals made of symmetrical pairs of tubulin-like protofilaments, which associate in an antiparallel array (similarly to the known Ca2+-induced sheets of FtsZ-His6). In contrast to the lateral interactions of microtubule protofilaments, we propose that the primary assembly product of FtsZ is the double-stranded filament, one or several of which might form the dynamic Z ring during prokaryotic cell division.

Archaea↗
PubMed2003

Energetics of the cooperative assembly of cell division protein FtsZ and the nucleotide hydrolysis switch.

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 +/- 100 cal mol-1 K-1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 degrees C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol-1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.

Bacterial Proteins↗
PubMed2004

Mechanism of 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase, a key enzyme in the methanopterin biosynthetic pathway.

The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, beta-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P), CO(2), and inorganic pyrophosphate (PP(i)). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO(2), then beta-RFA-P, and finally PP. The Michaelis parameters are as follows: K(m)pABA, 0.15 mm; K(m)PRPP, 1.50 mm; V(max), 375 nmol/min/mg; k(cat), 0.23 s(-1). CO(2) showed uncompetitive inhibition, K(i) = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K(i) = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, K(i) = 0.300 mm, under saturated PRPP and varied pABA. PP(i) exhibits competitive inhibition, K(i) = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 0.60 mm and K(2) = 1.900 mm, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.

Catalysis↗