Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii.

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

Structural Analysis of Glycine Sarcosine N-methyltransferase from Methanohalophilus portucalensis Reveals Mechanistic Insights into the Regulation of Methyltransferase Activity.

Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways' end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved.

A modulator domain controlling thermal stability in the Group II chaperonins of Archaea.

Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122°C. The C-terminal 15-25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif ((528)EKEKEKEGEK5(37)) proved to be a key domain for stabilization at or near 100°C. Mutations "tuned" the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca(2+), especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca(2+). The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns.

Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer.

Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part II: the effect of different methylated growth substrates.

Methanococcoides burtonii is a cold-adapted methanogenic archaeon from Ace Lake in Antarctica. Methanol and methylamines are the only substrates it can use for carbon and energy. We carried out quantitative proteomics using iTRAQ of M. burtonii cells grown on different substrates (methanol in defined media or trimethylamine in complex media), using techniques that enriched for secreted and membrane proteins in addition to cytoplasmic proteins. By integrating proteomic data with the complete, manually annotated genome sequence of M. burtonii, we were able to gain new insight into methylotrophic metabolism and the effects of methanol on the cell. Metabolic processing of methanol and methylamines is initiated by methyltransferases specific for each substrate, with multiple paralogs for each of the methyltransferases (similar to other members of the Methanosarcinaceae). In M. burtonii, most methyltransferases appear to have distinct roles in the metabolism of methylated substrates, although two methylamine methyltransferases appear to be nonfunctional. One set of methyltransferases for trimethylamine catabolism appears to be membrane associated, potentially providing a mechanism to directly couple trimethylamine uptake to demethylation. Important roles were highlighted for citrate synthase, glutamine synthetase, acetyl-CoA decarbonylase/synthase, and pyruvate synthase in carbon and nitrogen metabolism during growth on methanol. M. burtonii had only a marginal response to the provision of exogenous amino acids (from yeast extract), indicating that it is predisposed to the endogenous synthesis of amino acids. Growth on methanol appeared to cause oxidative stress in the cell, possibly through the formation of reactive nonoxygen species and formaldehyde, and the oxidative inactivation of corrinoid proteins, with the cell responding by elevating the synthesis of universal stress (Usp) proteins, several nucleic acid binding proteins, and a serpin. In addition, changes in levels of cell envelope proteins were linked to counteracting the disruptive solvent effects of methanol on cell membranes. This is the first global proteomic study to examine the effects of different carbon sources on the growth of an obligately methylotrophic methanogen.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part I: the effect of growth temperature.

The response of the cold-adapted (psychrophilic) methanogenic archaeon Methanococcoides burtonii to growth temperature was investigated using differential proteomics (postincorporation isobaric labeling) and tandem liquid chromatography-mass spectrometry (LC/LC-MS/MS). This is the first proteomic study of M. burtonii to include techniques that specifically enrich for both surface and membrane proteins and to assess the effects of growth temperature (4 vs 23 degrees C) and carbon source (trimethylamine vs methanol) on cellular protein levels. Numerous surface layer proteins were more abundant at 4 degrees C, indicating an extensive remodeling of the cell envelope in response to low temperature. Many of these surface proteins contain domains associated with cell adhesion. Within the cell, small proteins each composed of a single TRAM domain were recovered as important cold adaptation proteins and might serve as RNA chaperones, in an analogous manner to Csp proteins (absent from M. burtonii). Other proteins that had higher abundances at 4 degrees C can be similarly tied to relieving or resolving the adverse affects of cold growth temperature on translational capacity and correct protein folding. The proteome of M. burtonii grown at 23 degrees C was dominated by oxidative stress proteins, as well as a large number of integral membrane proteins of unknown function. This is the first truly global proteomic study of a psychrophilic archaeon and greatly expands knowledge of the cellular mechanisms underpinning cold adaptation in the Archaea.

Recombinant production, crystallization and preliminary X-ray analysis of PCNA from the psychrophilic archaeon Methanococcoides burtonii DSM 6242.

Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3 kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5 angstrom. A singleMbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40 angstrom resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained.

Proteomic and computational analysis of secreted proteins with type I signal peptides from the Antarctic archaeon Methanococcoides burtonii.

LC-MS/MS was used to identify secreted proteins in the Antarctic archaeon Methanococcoides burtonii. Seven proteins possessing a classical class 1 signal peptide were identified in the supernatant from cultures grown at 4 and 23 degrees C. The proteins included a putative S-layer cell surface protein, cell surface protein involved with cell adhesion, and trypsin-like serine protease. Protease activity was detected in the secreted fraction, and the signal peptide cleavage site of the protease was confirmed using Edman sequencing. The expression profile of putative cell surface proteins suggests a requirement for cell interactions during growth at low temperature. Sequences of the secreted proteins were used to compile a dataset containing a further 32 predicted secreted proteins from the Methanosarcinaceae. Many of these proteins were also S-layer cell surface proteins with a variety of predicted roles, particularly in cell-cell interaction. Computational analysis of signal peptides revealed a preference for lysine in the n-region, leucine in the h-region, and a eucaryal-type cleavage site, highlighting the mosaic nature of signal peptides in Archaea. This is the first study to experimentally characterize secreted proteins from a cold-adapted archaeon and provides new insight and a functional dataset for studying secretion in Archaea.

Methanosaeta harundinacea sp. nov., a novel acetate-scavenging methanogen isolated from a UASB reactor.

Two methanogenic strains, 8AcT and 6Ac, were isolated from an upflow anaerobic sludge blanket reactor treating beer-manufacture wastewater in Beijing, China. Cells of strains 8AcT and 6Ac were rod-shaped (0.8-1.0 x 3-5 microm) and non-motile, occurring singly or in pairs; however, at high cell density the cells were arranged in long chains within a common sheath. The two strains used acetate exclusively for growth and methane production. The specific growth rate of strain 8AcT was 0.030 h(-1) when growing in acetate (20 mM) at 37 degrees C. The temperature range for growth was 25-45 degrees C, with the fastest growth at 34-37 degrees C. The pH range for growth and methane production was 6.5-9.0, with the fastest growth at pH 7.2-7.6. The G+C content of genomic DNA of strain 8AcT was 55.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that the novel strains clustered with Methanosaeta species; the 16S rRNA gene sequence similarities between strain 8AcT and Methanosaeta concilii DSM 3013 and 'Methanosaeta thermophila' DSM 6194 were 92.5 and 87.3 %, respectively. The sequence similarity levels of mcrA, the gene encoding the alpha-subunit of methyl-coenzyme M reductase, and of the deduced amino acids of mcrA, between strain 8AcT and Methanosaeta concilii DSM 3671T were 36 and 78.9 %, respectively. Based on the phylogenetic and phenotypic analyses, the novel species Methanosaeta harundinacea sp. nov. is proposed, with strain 8AcT (= JCM 13211T = CGMCC 1.5026T) as the type strain.

Pyrrolysine and selenocysteine use dissimilar decoding strategies.

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies.

Effect of temperature on stability and activity of elongation factor 2 proteins from Antarctic and thermophilic methanogens.

Despite the presence and abundance of archaea in low-temperature environments, little information is available regarding their physiological and biochemical properties. In order to investigate the adaptation of archaeal proteins to low temperatures, we purified and characterized the elongation factor 2 (EF-2) protein from the Antarctic methanogen Methanococcoides burtonii, which was expressed in Escherichia coli, and compared it to the recombinant EF-2 protein from a phylogenetically related thermophile, Methanosarcina thermophila. Using differential scanning calorimetry to assess protein stability and enzyme assays for the intrinsic GTPase activity, we identified biochemical and biophysical properties that are characteristic of the cold-adapted protein. This includes a higher activity at low temperatures caused by a decrease of the activation energy necessary for GTP hydrolysis and a decreased activation energy for the irreversible denaturation of the protein, which indicates a less thermostable structure. Comparison of the in vitro properties of the proteins with the temperature-dependent characteristics of growth of the organisms indicates that additional cytoplasmic factors are likely to be important for the complete thermal adaptation of the proteins in vivo. This is the first study to address thermal adaptation of proteins from a free-living, cold-adapted archaeon, and our results indicate that the ability of the Antarctic methanogen to adapt to the cold is likely to involve protein structural changes.