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PubMed2003

Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel.

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.

Amino Acid Sequence↗
PubMed2003

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Amino Acid Sequence↗
PubMed2004

Selenocysteine tRNA-specific elongation factor SelB is a structural chimaera of elongation and initiation factors.

In all three kingdoms of life, SelB is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a UGA stop codon in the presence of a downstream mRNA hairpin loop. Here, we present the X-ray structures of SelB from the archaeon Methanococcus maripaludis in the apo-, GDP- and GppNHp-bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl-binding pocket. All three SelB structures reveal an EF-Tu:GTP-like domain arrangement. Upon binding of the GTP analogue GppNHp, a conformational change of the Switch 2 region in the GTPase domain leads to the exposure of SelB residues involved in clamping the 5' phosphate of the tRNA. A conserved extended loop in domain III of SelB may be responsible for specific interactions with tRNA(Sec) and act as a ruler for measuring the extra long acceptor arm. Domain IV of SelB adopts a beta barrel fold and is flexibly tethered to domain III. The overall domain arrangement of SelB resembles a 'chalice' observed so far only for initiation factor IF2/eIF5B. In our model of SelB bound to the ribosome, domain IV points towards the 3' mRNA entrance cleft ready to interact with the downstream secondary structure element.

Amino Acid Sequence↗
PubMed2007

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake.

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.

Adenosine Triphosphate↗
PubMed2006

Comparison of spectral counting and metabolic stable isotope labeling for use with quantitative microbial proteomics.

Spectral counting, a promising method for quantifying relative changes in protein abundance in mass spectrometry-based proteomic analysis, was compared to metabolic stable isotope labeling using (15)N/(14)N "heavy/light" peptide pairs. The data were drawn primarily from a Methanococcus maripaludis experiment comparing a wild-type strain with a mutant deficient in a key enzyme relevant to energy metabolism. The dataset contained both proteome and transcriptome measurements. The normalization technique used previously for the isotopic measurements was inappropriate for spectral counting, but a simple adjustment for sampling frequency was sufficient for normalization. This adjustment was satisfactory both for M. maripaludis, an organism that showed relatively little expression change between the wild-type and mutant strains, and Porphyromonas gingivalis, an intracellular pathogen that has demonstrated widespread changes between intracellular and extracellular conditions. Spectral counting showed lower overall sensitivity defined in terms of detecting a two-fold change in protein expression, and in order to achieve the same level of quantitative proteome coverage as the stable isotope method, it would have required approximately doubling the number of mass spectra collected.

Animals↗
PubMed2001

Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase. A member of a new class of Mg(2+)-dependent acid phosphatases.

Coenzyme M (CoM; 2-mercaptoethanesulfonic acid) is the terminal methyl carrier in methanogenesis. Methanogenic archaea begin the production of this essential cofactor by sulfonating phosphoenolpyruvate to form 2-phospho-3-sulfolactate. After dephosphorylation, this precursor is oxidized, decarboxylated and then reductively thiolated to form CoM. A thermostable phosphosulfolactate phosphohydrolase (EC 3.1.3.-) catalyzing the second step in CoM biosynthesis, was identified in the hyperthermophilic euryarchaeon Methanococcus jannaschii. The predicted ORF MJ1140 in the genome of M. jannaschii encodes ComB, a Mg2+-dependent acid phosphatase that is specific for 2-hydroxycarboxylic acid phosphate esters. Recombinantly expressed purified ComB efficiently hydrolyzes rac-2-phosphosulfolactate, (S)-2-phospholactate, phosphoglycolate and both enantiomers of 2-phosphomalate. In contrast to previously studied phosphoglycolate phosphatases, ComB has a low pH optimum for activity, a narrow substrate specificity and an amino acid sequence dissimilar to any biochemically characterized protein. Like other phosphatases that function via covalent phosphoenzyme intermediates, ComB can catalyze a transphosphorylation reaction. Homologs of comB are identified in all available cyanobacterial genome sequences and in genomes from phylogenetically diverse bacteria and archaea; most of these organisms lack homologs of other CoM biosynthetic genes. The broad and disparate distribution of comB homologs suggests that the gene has been recruited frequently into new metabolic pathways.

Acid Phosphatase↗
PubMed1997

Comparison of archaeal and bacterial genomes: computer analysis of protein sequences predicts novel functions and suggests a chimeric origin for the archaea.

Protein sequences encoded in three complete bacterial genomes, those of Haemophilus influenzae, Mycoplasma genitalium and Synechocystis sp., and the first available archaeal genome sequence, that of Methanococcus jannaschii, were analysed using the BLAST2 algorithm and methods for amino acid motif detection. Between 75% and 90% of the predicted proteins encoded in each of the bacterial genomes and 73% of the M. jannaschii proteins showed significant sequence similarity to proteins from other species. The fraction of bacterial and archaeal proteins containing regions conserved over long phylogenetic distances is nearly the same and close to 70%. Functions of 70-85% of the bacterial proteins and about 70% of the archaeal proteins were predicted with varying precision. This contrasts with the previous report that more than half of the archaeal proteins have no homologues and shows that, with more sensitive methods and detailed analysis of conserved motifs, archaeal genomes become as amenable to meaningful interpretation by computer as bacterial genomes. The analysis of conserved motifs resulted in the prediction of a number of previously undetected functions of bacterial and archaeal proteins and in the identification of novel protein families. In spite of the generally high conservation of protein sequences, orthologues of 25% or less of the M. jannaschii genes were detected in each individual completely sequenced genome, supporting the uniqueness of archaea as a distinct domain of life. About 53% of the M. jannaschii proteins belong to families of paralogues, a fraction similar to that in bacteria with larger genomes, such as Synechocystis sp. and Escherichia coli, but higher than that in H. influenzae, which has approximately the same number of genes as M. jannaschii. Certain groups of proteins, e.g. molecular chaperones and DNA repair enzymes, thought to be ubiquitous and represented in the minimal gene set derived by bacterial genome comparison, are missing in M. jannaschii, indicating massive non-orthologous displacement of genes responsible for essential functions. An unexpectedly large fraction of the M. jannaschii gene products, 44%, shows significantly higher similarity to bacterial than to eukaryotic proteins, compared with 13% that have eukaryotic proteins as their closest homologues (the rest of the proteins show approximately the same level of similarity to bacterial and eukaryotic homologues or have no homologues). Proteins involved in translation, transcription, replication and protein secretion are most closely related to eukaryotic proteins, whereas metabolic enzymes, metabolite uptake systems, enzymes for cell wall biosynthesis and many uncharacterized proteins appear to be 'bacterial'. A similar prevalence of proteins of apparent bacterial origin was observed among the currently available sequences from the distantly related archaeal genus, Sulfolobus. It is likely that the evolution of archaea included at least one major merger between ancestral cells from the bacterial lineage and the lineage leading to the eukaryotic nucleocytoplasm.

Algorithms↗
PubMed2000

S-adenosylmethionine decarboxylase of Bacillus subtilis is closely related to archaebacterial counterparts.

Bacillus subtilis synthesizes polyamines by decarboxylating arginine to agmatine, which is subsequently hydrolysed to putrescine. Spermidine is synthesized from putrescine and decarboxylated S-adenosylmethionine (dAdoMet). In Gram-negative bacteria and in eukaryotes, AdoMet is decarboxylated by an unusual 'pyruvoyl' AdoMet decarboxylase (SpeD), the catalytic pyruvoyl moiety of which is generated by serinolysis of an internal serine with self-cleavage of the protein at the upstream peptide bond. Neither the Gram-positive bacterial nor the archaeal counterpart of the Escherichia coli SpeD enzyme were known. We have identified the corresponding B. subtilis speD gene (formely ytcF). Heterologous expression of the cognate Methanococcus jannaschii protein, MJ0315, demonstrated that it displays the same activity as B. subtilis SpeD, indicating that spermidine biosynthesis in Gram-positive bacteria and in archaea follows a pathway very similar to that of Gram-negatives and eukarya. In B. subtilis, transcription of speD is modulated by spermidine and methionine. Its expression is high under usual growth conditions. In contrast, the SpeD protein self-cleaves slowly in vitro, a noticeable difference with its archaeal counterpart. Under certain growth conditions (minimal medium containing succinate and glutamate as a carbon source), speD is co-transcribed with gapB, the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme required for gluconeogenesis. This observation may couple polyamine metabolism to sulphur and carbon metabolism by a so far unknown mechanism.

Adenosylmethionine Decarboxylase↗
PubMed2004

Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS.

For more complete characterization of DNA-predicted proteins (including their posttranslational modifications) a "top-down" approach using high-resolution tandem MS is forwarded here by its application to methanogens in both hypothesis-driven and discovery modes, with the latter dependent on new automation benchmarks for intact proteins. With proteins isolated from ribosomes and whole-cell lysates of Methanococcus jannaschii (approximately 1,800 genes) using a 2D protein fractionation method, 72 gene products were identified and characterized with 100% sequence coverage via automated fragmentation of intact protein ions in a custom quadrupole/Fourier transform hybrid mass spectrometer. Three incorrect start sites and two modifications were found, with one of each determined for MJ0556, a 20-kDa protein with an unknown methylation at approximately 50% occupancy in stationary phase cells. The separation approach combined with the quadrupole/Fourier transform hybrid mass spectrometer allowed targeted and efficient comparison of histones from M. jannaschii, Methanosarcina acetivorans (largest Archaeal genome, 5.8 Mb), and yeast. This finding revealed a striking difference in the posttranslational regulation of DNA packaging in Eukarya vs. the Archaea. This study illustrates a significant evolutionary step for the MS tools available for characterization of WT proteins from complex proteomes without proteolysis.

Amino Acid Sequence↗
PubMed2004

An enzymatic molten globule: efficient coupling of folding and catalysis.

A highly active, monomeric chorismate mutase, obtained by topological redesign of a dimeric helical bundle enzyme from Methanococcus jannaschii, was investigated by NMR and various other biochemical techniques, including H/D exchange. Although structural disorder is generally considered to be incompatible with efficient catalysis, the monomer, unlike its natural counterpart, unexpectedly possesses all of the characteristics of a molten globule. Global conformational ordering, observed upon binding of a transition state analog, indicates that folding can be coupled to catalysis with minimal energetic penalty. These results support the suggestion that many modern enzymes might have evolved from molten globule precursors. Insofar as their structural plasticity confers relaxed substrate specificity and/or catalytic promiscuity, molten globules may also be attractive starting points for the evolution of new catalysts in the laboratory.

Chorismate Mutase↗
PubMed2005

Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications.

Type I restriction-modification enzymes are differentiated from type II and type III enzymes by their recognition of two specific dsDNA sequences separated by a given spacer and cleaving DNA randomly away from the recognition sites. They are oligomeric proteins formed by three subunits: a specificity subunit, a methylation subunit, and a restriction subunit. We solved the crystal structure of a specificity subunit from Methanococcus jannaschii at 2.4-A resolution. Two highly conserved regions (CRs) in the middle and at the C terminus form a coiled-coil of long antiparallel alpha-helices. Two target recognition domains form globular structures with almost identical topologies and two separate DNA binding clefts with a modeled DNA helix axis positioned across the CR helices. The structure suggests that the coiled-coil CRs act as a molecular ruler for the separation between two recognized DNA sequences. Furthermore, the relative orientation of the two DNA binding clefts suggests kinking of bound dsDNA and exposing of target adenines from the recognized DNA sequences.

Amino Acid Sequence↗
PubMed2005

An expanding family of archaeal transcriptional activators.

Transcriptional regulation in the archaea involves a mosaic of DNA-binding proteins frequently (although not exclusively) of bacterial type, modulating a eukaryal-type core transcription apparatus. Methanocaldococcus jannaschii (Mja) Ptr2, a homologue of the Lrp/AsnC family of bacterial transcription regulators that are among the most widely disseminated archaeal DNA-binding proteins, has been shown to activate transcription by its conjugate hyperthermophilic RNA polymerase. Here, two in vitro systems have been exploited to show that Ptr2 and a Lrp homologue from the thermophile Methanothermococcus thermolithotrophicus (Mth) activate transcription over a approximately 40 degrees C range, in conjunction with their cognate TATA-binding proteins (TBPs) and with heterologous TBPs. A closely related homologue from the mesophile Methanococcus maripaludis (Mma) is nearly inert as a transcriptional activator, but a cluster of mutations that converts a surface patch of Mma Lrp to identity with Ptr2 confers transcriptional activity. Mja, Mth, and Mma TBPs are interchangeable for basal transcription, but their ability to support Lrp-mediated transcriptional activation varies widely, with Mja TBP the most active and Mth TBP the least active partner. The implications of this finding for understanding the roles of TBP paralogues in supporting the gene-regulatory repertoires of archaeal genomes are briefly noted.

Amino Acid Sequence↗
PubMed2006

Structural plasticity of an aminoacyl-tRNA synthetase active site.

Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substrate-bound WT synthetase, as well as a mutant synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the alpha8-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyl-tRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.

Alanine↗
PubMed2006

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase.

Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifI(1) and nifI(2), which encode members of the PII family of nitrogen-regulatory proteins. Previous work demonstrated that nitrogenase activity in cell extracts was inhibited in the presence of NifI(1) and NifI(2), and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the NifI proteins in switch-off, we found proteins that interact with NifI(1) and NifI(2) and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI(2) resulted in copurification of NifI(1) and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI(2) prevented copurification of dinitrogenase but did not affect copurification of NifI(1). Similar results were obtained with His-tagged NifI(1). Gel-filtration chromatography demonstrated an interaction between purified NifI(1,2) and dinitrogenase that was inhibited by 2OG. The NifI proteins themselves formed a complex of approximately 85 kDa, which appeared to further oligomerize in the presence of 2OG. NifI(1,2) inhibited activity of purified nitrogenase when present in a 1:1 molar ratio to dinitrogenase, and 2OG fully relieved this inhibition. These results suggest a model for switch-off of nitrogenase activity, where direct interaction of a NifI(1,2) complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifI(1) and nifI(2) in the nif operons of all nitrogen-fixing Archaea and some anaerobic Bacteria suggests that this mode of nitrogenase regulation may operate in a wide variety of diazotrophs.

Archaeal Proteins↗
PubMed2003

Activation of archaeal transcription by recruitment of the TATA-binding protein.

The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, that are members of the Lrp/AsnC family of bacterial transcription regulators. In contrast, this archaeon's RNA polymerase and core transcription factors are of eukaryotic type. Using the M. jannaschii high-temperature in vitro transcription system, we show that Ptr2 is a potent transcriptional activator, and that it conveys its stimulatory effects on its cognate eukaryal-type transcription machinery from an upstream activating region composed of two Ptr2-binding sites. Transcriptional activation is generated, at least in part, by Ptr2-mediated recruitment of the TATA-binding protein to the promoter.

Base Sequence↗
PubMed2002

Noncoding RNA genes identified in AT-rich hyperthermophiles.

Noncoding RNA (ncRNA) genes that produce functional RNAs instead of encoding proteins seem to be somewhat more prevalent than previously thought. However, estimating their number and importance is difficult because systematic identification of ncRNA genes remains challenging. Here, we exploit a strong, surprising DNA composition bias in genomes of some hyperthermophilic organisms: simply screening for GC-rich regions in the AT-rich Methanococcus jannaschii and Pyrococcus furiosus genomes efficiently detects both known and new RNA genes with a high degree of secondary structure. A separate screen based on comparative analysis also successfully identifies noncoding RNA genes in P. furiosus. Nine of the 30 new candidate genes predicted by these screens have been verified to produce discrete, apparently noncoding transcripts with sizes ranging from 97 to 277 nucleotides.

Adenine↗
PubMed2001

BeF(3)(-) acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF(3)(-) complex with phosphoserine phosphatase.

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF(3)(-), which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-A resolution the x-ray crystal structure of the complex of BeF(3)(-) with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF(3)(-) is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg(2+), and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF(3)(-) x PSP and BeF(3)(-) x CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF(3)(-) for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Aspartic Acid↗
PubMed2003

Glutathione synthetase homologs encode alpha-L-glutamate ligases for methanogenic coenzyme F420 and tetrahydrosarcinapterin biosyntheses.

Proteins in the ATP-grasp superfamily of amide bond-forming ligases have evolved to function in a number of unrelated biosynthetic pathways. Previously identified homologs encoding glutathione synthetase, d-alanine:d-alanine ligase and the bacterial ribosomal protein S6:glutamate ligase have been vertically inherited within certain organismal lineages. Although members of this specificity-diverse superfamily share a common reaction mechanism, the nonoverlapping set of amino acid and peptide substrates recognized by each family provided few clues as to their evolutionary history. Two members of this family have been identified in the hyperthermophilic marine archaeon Methanococcus jannaschii and shown to catalyze the final reactions in two coenzyme biosynthetic pathways. The MJ0620 (mptN) locus encodes a tetrahydromethanopterin:alpha-l-glutamate ligase that forms tetrahydrosarcinapterin, a single carbon-carrying coenzyme. The MJ1001 (cofF) locus encodes a gamma-F420-2:alpha-l-glutamate ligase, which caps the gamma-glutamyl tail of the hydride carrier coenzyme F420. These two genes share a common ancestor with the ribosomal protein S6:glutamate ligase and a putative alpha-aminoadipate ligase, defining the first group of ATP-grasp enzymes with a shared amino acid substrate specificity. As in glutathione biosynthesis, two unrelated amino acid ligases catalyze sequential reactions in coenzyme F420 polyglutamate formation: a gamma-glutamyl ligase adds 1-3 l-glutamate residues and the ATP-grasp-type ligase described here caps the chain with a single alpha-linked l-glutamate residue. The analogous pathways for glutathione, F420, folate, and murein peptide biosyntheses illustrate convergent evolution of nonribosomal peptide biosynthesis through the recruitment of single-step amino acid ligases.

Adenosine Triphosphate↗