The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from Methanobrevibacter ruminantium

Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C₁ unit carrier where N⁵‐formyl‐tetrahydromethanopterin is converted to methenyl‐tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N‐terminal domain of six α‐helices encompassing a series of four β‐sheets and a predominantly anti‐parallel β–sheet at the C‐terminus flanked on one side by α‐helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation. Proteins 2013; 81:2064–2070. © 2013 Wiley Periodicals, Inc.     

The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: in vitro analysis of its chaperone activity

The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function. In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coili BL21 (DE3) cells. Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates. The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C.     

Transfer RNA genes from the hyperthermophilic Archaeon, Methanopyrus kandleri.

Genes encoding the Leu (GAG), Ser (UGA), Gln (UUG) and Lys (UUU) tRNAs have been cloned and sequenced from the deep sea hyperthermophilic Archaeon, Methanopyrus kandleri. Sequences conforming to the TATA box element established for methanogen promoters are located upstream of the tRNA(Gln) and tRNA(Lys) genes. All four of the tRNA genes appear to encode the 3' terminal CCA residues of the mature tRNA. These methanogen tRNAs are predicted to contain most, but not all, invariant residues and are characterized by a high level of G + C base pairing, consistent with the 98 degrees C optimum growth temperature of M. kandleri.     

Overproduction and one-step purification of the N 5,N 10-methenyltetrahydro-methanopterin cyclohydrolase (Mch) from the hyperthermophilic Methanopyrus kandleri

N ⁵,N ¹⁰-Methenyltetrahydromethanopterin cyclohydrolase (Mch) is an enzyme involved in methanogenesis from CO₂ and H₂ which represents the energy metabolism of Methanopyrus kandleri, a methanogenic Archaeon growing at a temperature optimum of 98°C. The gene mch from M. kandleri was cloned, sequenced, and expressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% in one step by chromatography on phenyl Sepharose in 80% ammonium sulfate. From 3.5 g cells (250 mg protein), approximately 18 mg cyclohydrolase was obtained. The purified enzyme showed essentially the same catalytic properties as the enzyme purified from M. kandleri cells. The primary structure and properties of the cyclohydrolase are compared with those of the enzyme from Methanococcus jannaschii (growth temperature optimum 85°C), from Methanobacterium thermoautotrophicum (65°C), and from Methanosarcina barkeri (37°C). Of the four enzymes, that from M. kandleri has the lowest isoelectric point (3.8) and the lowest hydrophobicity of amino acid composition. Besides, it has the highest relative content of glutamate, leucine, and valine and the lowest relative content of isoleucine, serine, and lysine. Some of these properties are unusual for enzymes from hyperthermophilic organisms. They may reflect the observation that the cyclohydrolase from M. kandleri is not only adapted to hyperthermophilic conditions but also to the high intracellular concentrations of lyotrophic salts prevailing in this organism. Received: July 14, 1997 / Accepted: August 28, 1997     

An ancestral nuclear protein assembly: crystal structure of the Methanopyrus kandleri histone

Eukaryotic histone proteins condense DNA into compact structures called nucleosomes. Nucleosomes were viewed as a distinguishing feature of eukaryotes prior to identification of histone orthologs in methanogens. Although evolutionarily distinct from methanogens, the methane-producing hyperthermophile Methanopyrus kandleri produces a novel, 154-residue histone (HMk). Amino acid sequence comparisons show that HMk differs from both methanogenic and eukaryotic histones, in that it contains two histone-fold ms within a single chain. The two HMk histone-fold ms, N and C terminal, are 28% identical in amino acid sequence to each other and approximately 21% identical in amino acid sequence to other histone proteins. Here we present the 1.37-A-resolution crystal structure of HMk and report that the HMk monomer structure is homologous to the eukaryotic histone heterodimers. In the crystal, HMk forms a dimer homologous to [H3-H4](2) in the eukaryotic nucleosome. Based on the spatial similarities to structural ms found in the eukaryotic nucleosome that are important for DNA-binding, we infer that the Methanopyrus histone binds DNA in a manner similar to the eukaryotic histone tetramer [H3-H4](2).     

Methanopyrus kandleri glutamyl-tRNA reductase.

The initial reaction of tetrapyrrole formation in archaea is catalyzed by a NADPH-dependent glutamyl-tRNA reductase (GluTR). The hemA gene encoding GluTR was cloned from the extremely thermophilic archaeon Methanopyrus kandleri and overexpressed in Escherichia coli. Purified recombinant GluTR is a tetrameric enzyme with a native M(r) = 190,000 +/- 10,000. Using a newly established enzyme assay, a specific activity of 0.75 nmol h(-1) mg(-1) at 56 degrees C with E. coli glutamyl-tRNA as substrate was measured. A temperature optimum of 90 degrees C and a pH optimum of 8.1 were determined. Neither heme cofactor, nor flavin, nor metal ions were required for GluTR catalysis. Heavy metal compounds, Zn(2+), and heme inhibited the enzyme. GluTR inhibition by the newly synthesized inhibitor glutamycin, whose structure is similar to the 3' end of the glutamyl-tRNA substrate, revealed the importance of an intact chemical bond between glutamate and tRNA(Glu) for substrate recognition. The absolute requirement for NADPH in the reaction of GluTR was demonstrated using four NADPH analogues. Chemical modification and site-directed mutagenesis studies indicated that a single cysteinyl residue and a single histidinyl residue were important for catalysis. It was concluded that during GluTR catalysis the highly reactive sulfhydryl group of Cys-48 acts as a nucleophile attacking the alpha-carbonyl group of tRNA-bound glutamate with the formation of an enzyme-localized thioester intermediate and the concomitant release of tRNA(Glu). In the presence of NADPH, direct hydride transfer to enzyme-bound glutamate, possibly facilitated by His-84, leads to glutamate-1-semialdehyde formation. In the absence of NADPH, a newly discovered esterase activity of GluTR hydrolyzes the highly reactive thioester of tRNA(Glu) to release glutamate.     

Activation and thermostabilization effects of cyclic 2,3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri

Enzymes involved in methane formation from carbon dioxide and dihydrogen in Methanopyrus kandleri require high concentrations (> 1 M) of lyotropic salts such as K₂HPO₄/KH₂PO₄ or (NH₄)₂SO₄ for activity and for thermostability. The requirement correlates with high intracellular concentrations of cyclic 2,3-diphosphoglycerate (cDPG; ≈ 1 M) in this hyperthermophilic organism. We report here on the effects of potassium cDPG on the activity and thermostability of the two methanogenic enzymes cyclohydrolase and formyltransferase and show that at cDPG concentrations prevailing in the cells the investigated enzymes are highly active and completely thermostable. At molar concentrations also the potassium salts of phosphate and of 2,3-bisphosphoglycerate, the biosynthetic precursor of cDPG, were found to confer activity and thermostability to the enzymes. Thermodynamic arguments are discussed as to why cDPG, rather than these salts, is present in high concentrations in the cells of Mp. kandleri. Received: 18 June 1998 / Accepted: 24 August 1998     

Lipid analysis of Methanopyrus kandleri

Abstract Non-polar and polar lipids were isolated from Methanopyrus kandleri . Non-polar lipids accounted for 50% w/w of total lipids, with a high proportion of 2,3-di- O -geranylgeranyl-sn-glycerol, 2,3-di- O -phytanyl-sn-glycerol and geranylgeraniol. The core lipids prepared by mild acid methanolysis consisted exclusively of 2,3-di- O -phytanyl-sn-glycerol. Two-dimensional TLC showed mostly glycolipids, and minor amounts of aminophospholipids, phosphoglycolipids and phospholipids. The purification yielded three diglycosyl-lipids (50% of total polar lipids), one triglycosyl-lipid (5%) and six glycosyl-lipids with five glycosyl-groups (36%), which consisted of glucose, galactose and mannose. The lipid analysis supports the unique position of Methanopyrus kandleri within the 16S rRNA-based phylogenetic tree.     

Structure and specificity of FEN‐1 from Methanopyrus kandleri

DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG‐like protein Mk0566 from M. kandleri, its biochemical activity and three‐dimensional structure were investigated. Both were found to be more similar to human FEN‐1 than human XPG, suggesting a biological role in replication and long‐patch base excision repair rather than in NER. Proteins 2015; 83:188–194. © 2014 Wiley Periodicals, Inc.     

Structural analysis by reductive cleavage with LiAlH4 of an allyl ether choline-phospholipid, archaetidylcholine, from the hyperthermophilic methanoarchaeon Methanopyrus kandleri

A choline-containing phospholipid (PL-4) in Methanopyrus kandleri cells was identified as archaetidylcholine, which has been described by Sprott et al. (1997). The PL-4 consisted of a variety of molecular species differing in hydrocarbon composition. Most of the PL-4 was acid-labile because of its allyl ether bond. The identity of PL-4 was confirmed by thin-layer chromatography followed by positive staining with Dragendorff reagent and fast-atom bombardment-mass spectrometry. A new method of LiAlH4 hydrogenolysis was developed to cleave allyl ether bonds and recover the corresponding hydrocarbons. We confirmed the validity of the LiAlH4 method in a study of the model compound synthetic unsaturated archaetidic acid (2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate). Saturated ether bonds were not cleaved by the LiAlH4 method. The hydrocarbons formed following LiAlH4 hydrogenolysis of PL-4 were identified by gas-liquid chromatography and mass spectrometry. Four kinds of hydrocarbons with one to four double bonds were detected: 47% of the hydrocarbons had four double bonds; 11% had three double bonds; 14% had two double bonds; 7% had one double bond; and 6% were saturated species. The molecular species composition of PL-4 was also estimated based on acid lability: 77% of the molecular species had two acid-labile hydrocarbons; 11% had one acid-labile and one acid-stable hydrocarbon; and 11% had two acid-stable hydrocarbons. To our knowledge, this is the first report of a specific chemical degradation method for the structural analysis of allyl ether phospholipid in archaea.     

A mutation affecting the association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri and its influence on the enzyme's activity and thermostability.

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunit of molecular mass 32 kDa. The enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. Oligomerization is required for enzyme activity and thermostability. We report here on a subunit interface mutation (R261E) which affects the dimer/tetramer part of the association equilibrium of formyltransferase. With the mutant protein it was shown that tetramerization is not required for activity but is necessary for high thermostability.     

The structure of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix

The structure of the recombinant medium chain alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Aeropyrum pernix has been solved by the multiple anomalous dispersion technique using the signal from the naturally occurring zinc ions. The enzyme is a tetramer with 222 point group symmetry. The ADH monomer is formed from a catalytic and a cofactor-binding domain, with the overall fold similar to previously solved ADH structures. The 1.62 A resolution A.pernix ADH structure is that of the holo form, with the cofactor NADH bound into the cleft between the two domains. The electron density found in the active site has been interpreted to be octanoic acid, which has been shown to be an inhibitor of the enzyme. This inhibitor is positioned with its carbonyl oxygen atom forming the fourth ligand of the catalytic zinc ion. The structural zinc ion of each monomer is present at only partial occupancy and in its absence a disulfide bond is formed. The enhanced thermal stability of the A.pernix ADH is thought to arise primarily from increased ionic and hydrophobic interactions on the subunit interfaces.     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83°C and 98°C, respectively. Both Archaea contain an active N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus.The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K₂HPO₄ and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K₂HPO₄ (1 M) for optimal thermostability at 90°C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K₂HPO₄ concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.Abbreviations H₄MPT tetrahydromethanopterin - MFR methanofuran - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₂H₄MPT N ⁵,N ¹⁰-methenyl-H₄MPT - CHO–H₄MPT N ⁵ formyl-H₄MPT - CHO-MFR formyl-MFR - cyclohydrolase N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase - MOPS 3-(N-morpholino) propane sulfonic acid - TRICINE N-tris (hydroxymethyl) methyl glycine - 1 U=1 mol/min     

X-ray structure of pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis.

BACKGROUND: Pyrrolidone carboxyl peptidases (pcps) are a group of exopeptidases responsible for the hydrolysis of N-terminal pyroglutamate residues from peptides and proteins. The bacterial and archaeal pcps are members of a conserved family of cysteine proteases. The pcp from the hyperthermophilic archaeon Thermococcus litoralis is more thermostable than the bacterial enzymes with which it has up to 40% sequence identity. The pcp activity in archaea and eubacteria is proposed to be involved in detoxification processes and in nutrient metabolism; eukaryotic counterparts of the enzyme are involved in the processing of biologically active peptides. RESULTS: The crystal structure of pcp has been determined by multiple isomorphous replacement techniques at 1.73 A resolution and refined to an R factor of 18.7% (Rfree = 21.4%). The enzyme is a homotetramer of single open alpha/beta domain subunits, with a prominent hydrophobic core formed from loops coming together from each monomer. The active-site residues have been identified as a Cys143-His167-Glu80 catalytic triad. Structural homology to enzymes of different specificity and mechanism has been identified. CONCLUSIONS: The Thermococcus pcp has no sequence or structural homology with other members of the cysteine protease family. It does, however, show considerable similarities to other hydrolytic enzymes of widely varying substrate specificity and mechanism, suggesting that they are the products of divergent evolution from a common ancestor. The enhanced thermostability of the T. litoralis pcp may arise from hydrophobic interactions between the subunits and the presence of intersubunit disulphide bridges.     

The cbiS gene of the archaeon Methanopyrus kandleri AV19 encodes a bifunctional enzyme with adenosylcobinamide amidohydrolase and alpha-ribazole-phosphate phosphatase activities

Here we report the initial biochemical characterization of the bifunctional alpha-ribazole-P (alpha-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AV19. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal alpha-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and alpha-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established alpha-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.     

The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses

Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).