A molecular phylogeny of thermophilic fungi

Sequences from 86 fungal genomes and from the two outgroup genomes Arabidopsis thaliana and Drosophila melanogaster were analyzed to construct a robust molecular phylogeny of thermophilic fungi, which are potentially rich sources of industrial enzymes. To provide experimental reference points, growth characteristics of 22 reported thermophilic or thermotolerant fungi, together with eight mesophilic species, were examined at four temperatures: 22 °C, 34 °C, 45 °C, and 55 °C. Based on the relative growth performances, species with a faster growth rate at 45 °C than at 34 °C were classified as thermophilic, and species with better or equally good growth at 34 °C compared to 45 °C as thermotolerant. We examined the phylogenetic relationships of a diverse range of fungi, including thermophilic and thermotolerant species, using concatenated amino acid sequences of marker genes mcm7, rpb1, and rpb2 obtained from genome sequencing projects. To further elucidate the phylogenetic relationships in the thermophile-rich orders Sordariales and Eurotiales, we used nucleotide sequences from the nuclear ribosomal small subunit (SSU), the 5.8S gene with internal transcribed spacers 1 and 2 (ITS 1 and 2), and the ribosomal large subunit (LSU) to include additional species for analysis. These phylogenetic analyses clarified the position of several thermophilic taxa. Thus, Myriococcum thermophilum and Scytalidium thermophilum fall into the Sordariales as members of the Chaetomiaceae, Thermomyces lanuginosus belongs to the Eurotiales, Malbranchea cinnamomea is a member of the Onygenales, and Calcarisporiella thermophila is assigned to the basal fungi close to the Mucorales. The mesophilic alkalophile Acremonium alcalophilum clusters with Verticillium albo-atrum and Verticillium dahliae, placing them in the recently established order Glomerellales. Taken together, these data indicate that the known thermophilic fungi are limited to the Sordariales, Eurotiales, and Onygenales in the Ascomycota and the Mucorales with possibly an additional order harbouring C. thermophila in the basal fungi. No supporting evidence was found for thermophilic species belonging to the Basidiomycota.     

Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpE_Tth) at 3.23 Å resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpE_Eco), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpE_Tth. In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpE_Eco structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpE_Tth correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpE_Tth causes functional deficiency of nucleotide exchange factor in response to the heat shock.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

The N-terminal region is crucial for the thermostability of the G-domain of Bacillus stearothermophilus EF-Tu

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability for the whole protein. Here we chose to locate the thermostability determinants distinguishing the thermophilic domain 1 from a mesophilic domain 1. By an approach of systematically swapping protein regions differing between G-domains from mesophilic Bacillus subtilis and thermophilic B. stearothermophilus, we demonstrate that a small portion of the protein, the N-terminal 12 amino acid residues, plays a key role in the thermostability of this domain. We suggest that the thermostabilizing effect of the N-terminal region could be mediated by stabilizing the functionally important effector region. Finally, we demonstrate that the effect of the N-terminal region is significant also for the thermostability of the full-length EF-Tu.     

Key cofactors of Photosystem II cores from four organisms identified by 1.7-K absorption, CD and MCD

Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98–103]. Electrochromic shifts resulting from Q_A reduction by 1.7-K illumination were recorded, and the Q_x and Q_y absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Q_x transition is ∼3 nm (100 cm⁻¹) to higher energy in cyanobacteria than in the plants. The predominant Q_y shift appears in the range 683–686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Q_y region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.     

Crystal structure of prephenate dehydrogenase from Streptococcus mutans

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD⁺ shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between β6 and β7, which may be a reason for the high K_m values of PDH from Streptococcus mutans.     

Thermophilic fungi: An assessment of their potential for growth in soil

An attempt has been made to forecast the potential of thermophilic fungi to grow in soil in the laboratory and in the field in the presence of a predominantly mesophilic fungal flora at usual temperature. The respiratory rate of thermophilic fungi was markedly responsive to changes in temperature, but that of mesophilic fungi was relatively independent of such changes. This suggested that in a thermally fluctuating environment, thermophilic fungi may be at a physiological disadvantage compared to mesophilic fungi. In mixed cultures in soil plates, thermophilic fungi outgrew mesophilic fungi under a fluctuating temperature regime only when the amplitude of the fluctuating temperatures was small and approached their temperature optima for growth. An antibody probe was used to detect the activity of native or an introduced strain of a thermophilic fungus,Thermomyces lanuginosus, under field conditions. The results suggest that although widespread, thermophilic fungi are ordinarily not an active component of soil microflora. Their presence in soil most likely may be the result of the aerial dissemination of propagules from composting plant material.     

Temperature-induced specific lipid desaturation in the thermophilic cyanobacterium Synechococcus vulcanus

Cyanobacteria desaturate fatty acids in the membrane lipids in response to decrease in temperature. We examined the changes in lipid and fatty acid composition in the thermophilic cyanobacterium Synechococcus vulcanus, which is characterized by an optimum growth temperature of 55°C. During temperature acclimation to 45°C or 35°C, the cells synthesized oleic acid at the expense of stearic acid in the membrane lipids. Unlike mesophilic cyanobacteria, S. vulcanus did not show any significant adaptive desaturation in the galactolipids monogalactosyl diacylglycerol and digalactosyl diacylglycerol, that comprise 50% and 30% of total membrane lipids, respectively. The major changes in fatty acid unsaturation were observed in the sulfolipid sulfoquinovosyl diacylglycerol.     

Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.     

Modulation of copper site properties by remote residues determines the stability of plastocyanins

The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environment is highly conserved in cyanobacterial plastocyanins. However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis. We have performed neutral amino-acid substitutions at loops of Phormidium plastocyanin far from the copper site. Notably, mutation P49G/G50P confers a redox-dependent thermal stability similar to that of the mesophilic plastocyanin. Moreover, X-ray absorption spectroscopy reveals that P49G/G50P mutation makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis plastocyanin.     

Structural dynamics of archaeal small heat shock proteins

Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.     

Mesophile versus thermophile: insights into the structural mechanisms of kinetic stability

Obtaining detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. Comparisons between proteins adapted to survive extreme temperatures with their mesophilic homologs are likely to provide valuable information on the interactions relevant to the unfolding transition. For kinetically stable proteins such as alpha-lytic protease (alphaLP) and its family members, their large free energy barrier to unfolding is central to their biological function. To gain new insights into the mechanisms that underlie kinetic stability, we have determined the structure and high temperature unfolding kinetics of a thermophilic homolog, Thermobifida fusca protease A (TFPA). These studies led to the identification of a specific structural element bridging the N and C-terminal domains of the protease (the "domain bridge") proposed to be associated with the enhanced high temperature kinetic stability in TFPA. Mutagenesis experiments exchanging the TFPA domain bridge into alphaLP validate this hypothesis and illustrate key structural details that contribute to TFPA's increased kinetic thermostability. These results lead to an updated model for the unfolding transition state structure for this important class of proteases in which domain bridge undocking and unfolding occurs at or before the TS. The domain bridge appears to be a structural element that can modulate the degree of kinetic stability of the different members of this class of proteases.     

Differential thermal effects on the energy distribution between photosystem II and photosystem I in thylakoid membranes of a psychrophilic and a mesophilic alga

Sensitivity of the photosynthetic thylakoid membranes to thermal stress was investigated in the psychrophilic Antarctic alga Chlamydomonas subcaudata. C. subcaudata thylakoids exhibited an elevated heat sensitivity as indicated by a temperature-induced rise in F_o fluorescence in comparison with the mesophilic species, Chlamydomonas reinhardtii. This was accompanied by a loss of structural stability of the photosystem (PS) II core complex and functional changes at the level of PSI in C. reinhardtii, but not in C. subcaudata. Lastly, C. subcaudata exhibited an increase in unsaturated fatty acid content of membrane lipids in combination with unique fatty acid species. The relationship between lipid unsaturation and the functioning of the photosynthetic apparatus under elevated temperatures is discussed.     

Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales

Cultivated members of the order Thermotogales comprise only thermophilic to hyperthermophilic anaerobic microorganisms. However, based on molecular studies, the existence of mesophilic members (“mesotoga”) within this order has been postulated but has not been demonstrated by cultural approaches so far. A “mesotoga” (strain PhosAc3) that belonged to an uncultivated lineage distantly related to the thermophilic Kosmotoga genus has now been cultivated in axenic culture. It grew between 30 °C and 50 °C (optimum 40 °C) and oxidized lactate using elemental sulphur as a terminal electron acceptor. Further genomic and physiological characterization of strain PhosAc3 will be important not only for understanding bacterial adaptation to high and moderate temperatures at small evolutionary scales, but also because “mesotoga” might play a crucial ecological role in ecosystems polluted by aromatic compounds.     

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA₁ or the psbA₃ gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491–1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b₅₅₉ (Cyt b₅₅₉) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b₅₅₉ properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b₅₅₉ likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.     

Structural studies show energy transfer within stabilized phycobilisomes independent of the mode of rod–core assembly

The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2–5 cylinders surrounded by 6–8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4 Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo.     

An analysis of temperature adaptation in cold active, mesophilic and thermophilic Bacillus α-amylases

A comparative biochemical and structural study was performed on a cold active α-amylase from Bacillus cereus (BCA) and two well-known homologous mesophilic and thermophilic α-amylases from Bacillus amyloliquefaciens (BAA) and Bacillus licheniformis (BLA). In spite of a high degree of sequence and structural similarity, drastic variations were found for T_opt as 50, 70 and 90 °C for BCA, BAA and BLA, respectively. The half-lives of thermoinactivation were 1 and 9 min for BCA and BAA at 80 °C respectively, whilst there was no inactivation for BLA at this temperature. Thermodynamic studies on inactivation process suggested that lower thermostability of BCA is due to lower inactivation slope of the Arrhenius plots and subsequently, lower E_a and ΔH^#. Increased K_m and accessible surface area for catalytic residues along with a decreased number of internal interactions in this region in BCA compared to BLA suggest that BCA substrate-binding site might be temperature sensitive and is probably more flexible. On the other hand, fewer ion pairs, destructive substitutions and disruption of aromatic interaction networks in structurally critical regions of Bacillus α-amylases result in a severe decrease in BCA thermostability compared to its mesophilic and thermophilic homologues.     

Heat shock protein HSP70B as a marker for genotype resistance to environmental stress in Chlorella species from contrasting habitats

The cellular response of three Chlorella species that differ in their temperature preferences and tolerance – Chlorella vulgaris — Antarctic, C. vulgaris strain 8/1 — thermophilic, and Chlorella kesslery — mesophilic – is analysed.     

Biochemical characterization and homology modeling of a purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus: Insights into mechanisms of protein stabilization

We report the biochemical and structural characterization of the purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus (SsIAG-NH). SsIAG-NH is a homodimer of 70 kDa specific for adenosine, guanosine and inosine. SsIAG-NH is highly thermophilic and is characterized by extreme thermodynamic stability (T_m, 107 °C), kinetic stability and remarkable resistance to guanidinium chloride-induced unfolding. A disulfide bond that, on the basis of SDS-PAGE is positioned intersubunits, plays an important role in thermal stability. SsIAG-NH shares 43% sequence identity with the homologous pyrimidine-specific nucleoside hydrolase from S. solfataricus (SsCU-NH). The comparative sequence alignment of SsIAG-NH, SsCU-NH, purine non-specific nucleoside hydrolase from Crithidia fasciculata and purine-specific nucleoside hydrolase from Trypanosoma vivax shows that, only few changes in the base pocket are responsible for different substrate specificity of two S. solfataricus enzymes. The structure of SsIAG-NH predicted by homology modeling allows us to infer the role of specific residues in substrate specificity and thermostability.     

"Cold spots" in protein cold adaptation: Insights from normalized atomic displacement parameters (B'-factors)

Many analyses published in the last decade suggest that enzymes isolated from cold-adapted organisms are characterized by a higher flexibility of their molecular structure. Recently, it has been argued that all cold-adapted enzymes with catalytic efficiency greater than that of their mesophilic counterparts display local flexibility or rigidity that are likely to cooperate, each acting on specific areas of the enzyme structure. Here we report an analysis of the normalized thermal B-factor distributions in psychrophilic proteins compared with those of their mesophilic and thermophilic counterparts with the aim to detect statistically significant local variations of relative backbone flexibility possibly linked to cold adaptation. We utilized a strategy based mainly on intra-family comparison of local distribution of normalized B-factors. After careful statistical treatment of data, the picture emerging from our results suggests that the distribution of the flexibility in psychrophilic enzymes is locally more heterogeneous than in their respective mesophilic homologues.