Characterization and Immunomodulatory Effects of High Molecular Weight Fucoidan Fraction from the Sporophyll of Undaria pinnatifida in Cyclophosphamide-Induced Immunosuppressed Mice.

Immunomodulation involves two mechanisms, immunostimulation and immunosuppression. It is a complex mechanism that regulates the pathophysiology and pathogenesis of various diseases affecting the immune system. Immunomodulators can be used as immunostimulators to reduce the side effects of drugs that induce immunosuppression. In this study, we characterized the chemical composition of high molecular weight fucoidan (HMWF) and low molecular weight fucoidan and compared their functions as natural killer (NK) cell-derived immunostimulators in vitro. We also tested the effectiveness of HMWF, which has a relatively high function in vitro, as an immunostimulator in immunosuppressed animal models. In these models, HWMF significantly restored NK cell cytotoxicity and granzyme B release to the control group level. In addition, the expression of interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α also increased in the spleen. This study suggests that HMWF acts as an effective immunostimulant under immunosuppressive conditions.

Structure and Regulation of the Movement of Human Myosin VIIA.

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca(2+). Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca(2+), and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.

Effect of nitric oxide on conformational changes of ovalbumin accompanying self-assembly into non-disease-associated fibrils.

Ovalbumin (OVA), one of the members of the serpin-superfamily, is the major protein in chicken eggs. Many studies have demonstrated the polymerization ability of OVA but the detailed molecular mechanisms demonstrating its conformational changes accompanying fibril formation are still unclear. This study revealed nitric oxide (NO) induced conformational changes and oligomerization of egg white OVA, resulting in polymerized fibrils. Electron microscopic analysis showed that NO treatment to OVA under mild acidic condition resulted in morphological changes, producing structures similar to the long protein fibrils found in egg white. Spectroscopic analysis and mass spectrometry found that NO-treated OVA contains increased number of ß-sheet, indicating transition from α-helixes to ß-sheets, and S-nitrosylation of OVA cysteine residue 367. Structural modeling showed that S-nitrosocysteine, Cys367NO, is located in the amyloidogenic core region of the C-terminal region, nearby the N-terminal core region where the α-to-ß transition is induced. Such results provide a potential mechanism for non-disease-associated fibril formation of OVA.

Rational design of a glycosynthase by the crystal structure of ß-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures.

The crystal structure of ß-galactosidase from Bacillus circulans (BgaC) was determined at 1.8Å resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-ß domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae ß(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from α-d-galactopyranosyl fluoride (αGalF) to different ß-linked N-acetylglucosamine acceptor substrates in good yield (40-90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of ß1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.

Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1.

RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNase H1) requires ⩾2M NaCl, ⩾10mM MnCl2, or ⩾300mM MgCl2 for folding. To understand the structural basis for this salt-dependent folding of Halo-RNase H1, the crystal structure of Halo-RNase H1 was determined in the presence of 10mM MnCl2. The structure of Halo-RNase H1 highly resembles those of metagenome-derived LC11-RNase H1 and Sulfolobus tokodaii RNase H1 (Sto-RNase H1), except that it contains two Mn(2+) ions at the active site and has three bi-aspartate sites on its surface. To examine whether negative charge repulsion at these sites are responsible for low-salt denaturation of Halo-RNase H1, a series of the mutant proteins of Halo-RNase H1 at these sites were constructed. The far-UV CD spectra of these mutant proteins measured in the presence of various concentrations of NaCl suggest that these mutant proteins exist in an equilibrium between a partially folded state and a folded state. However, the fraction of the protein in a folded state is nearly 0% for the active site mutant, 40% for the bi-aspartate site mutant, and 70% for the mutant at both sites in the absence of salt. The active site mutant requires relatively low concentration (∼0.5M) of salt for folding. These results suggest that suppression of negative charge repulsion at both active and bi-aspartate sites by salt is necessary to yield a folded protein.

Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.

The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.

Molecular assembly of thyroglobulin induced by in vitro nitric oxide treatments: implication its role in thyroid cells.

The formation of amyloid-like fibrils, which polymerize from various soluble proteins under physiological and acidic conditions, causes a wide range of protein-folding diseases, such as Alzheimer's disease and Parkinson's disease. Fibril assembly in in vitro solutions containing nitric oxide, a free radical that functions as an important signalling molecule involved in numerous physiological and pathological processes, has not been reported. Here, we investigated the protein assembly that occur in thyroglobulin under mildly acidic conditions in the presence of nitric oxide. Solution studies, size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation, demonstrated the size changes of thyroglobulin oligomers after nitric oxide treatment. Following electron microscopic analysis visualized their structural changes and revealed that the molecules can morphologically form polymerized fibril assemblies with a length of 2-5 µm and width 10-100 nm. Taken together, these results provide suggestive evidence for the propensity of forming polymerized thyroglobulin fibrils implying their presence in thyroid cells, which may be related to the onset or progression of thyroid diseases.

Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain.

Tk-subtilisin (Gly70-Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk-subtilisin is produced from its inactive precursor, Pro-Tk-subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide. Tk-propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar-to-polar amino acid substitution at this core affects the activation rate of Pro-Tk-subtilisin, the Pro-Tk-subtilisin derivative with the Phe17 → His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) were constructed. The crystal structure of Pro-F17H/S324A was nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-subtilisin. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C were also nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). However, the activation rate of Pro-F17H greatly increased when compared with that of Pro-Tk-subtilisin, such that Pro-F17H is efficiently activated even at 40°C. The far-UV circular dichroism spectrum of F17H-propeptide did not exhibit a broad trough at 205-230 nm, which is observed in the spectrum of Tk-propeptide. F17H-propeptide is more susceptible to chymotryptic degradation than Tk-propeptide. These results suggest that F17H-propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk-subtilisin. Thus, destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-subtilisin.

Structural insights into the regulation of sialic acid catabolism by the Vibrio vulnificus transcriptional repressor NanR.

Pathogenic and commensal bacteria that experience limited nutrient availability in their host have evolved sophisticated systems to catabolize the mucin sugar N-acetylneuraminic acid, thereby facilitating their survival and colonization. The correct function of the associated catabolic machinery is particularly crucial for the pathogenesis of enteropathogenic bacteria during infection, although the molecular mechanisms involved with the regulation of the catabolic machinery are unknown. This study reports the complex structure of NanR, a repressor of the N-acetylneuraminate (nan) genes responsible for N-acetylneuraminic acid catabolism, and its regulatory ligand, N-acetylmannosamine 6-phosphate (ManNAc-6P), in the human pathogenic bacterium Vibrio vulnificus. Structural studies combined with electron microscopic, biochemical, and in vivo analysis demonstrated that NanR forms a dimer in which the two monomers create an arched tunnel-like DNA-binding space, which contains positively charged residues that interact with the nan promoter. The interaction between the NanR dimer and DNA is alleviated by the ManNAc-6P-mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of the nan genes. Survival studies in which mice were challenged with a ManNAc-6P-binding-defective mutant strain of V. vulnificus demonstrated that this relocation of NanR residues is critical for V. vulnificus pathogenesis. In summary, this study presents a model of the mechanism that regulates sialic acid catabolism via NanR in V. vulnificus.

Double-stranded RNA of intestinal commensal but not pathogenic bacteria triggers production of protective interferon-ß.

The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor by using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered interferon-ß (IFN-ß) production, which protected mice from experimental colitis. The LAB-induced IFN-ß response was diminished by dsRNA digestion and treatment with endosomal inhibitors. Pathogenic bacteria contained less dsRNA and induced much less IFN-ß than LAB, and dsRNA was not involved in pathogen-induced IFN-ß induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and suggest that dsRNA in commensal bacteria contributes to anti-inflammatory and protective immune responses.

Crystal structure of metagenome-derived LC9-RNase H1 with atypical DEDN active site motif.

The crystal structure of metagenome-derived LC9-RNase H1 was determined. The structure-based mutational analyses indicated that the active site motif of LC9-RNase H1 is altered from DEDD to DEDN. In this motif, the location of the second glutamate residue is moved from αA-helix to ß1-strand immediately next to the first aspartate residue, as in the active site of RNase H2. However, the structure and enzymatic properties of LC9-RNase H1 highly resemble those of RNase H1, instead of RNase H2. We propose that LC9-RNase H1 represents bacterial RNases H1 with an atypical DEDN active site motif, which are evolutionarily distinct from those with a typical DEDD active site motif.

Crystal structure of metagenome-derived LC11-RNase H1 in complex with RNA/DNA hybrid.

LC11-RNase H1 is a Sulfolobus tokodaii RNase H1 (Sto-RNase H1) homologue isolated by metagenomic approach. In this study, the crystal structure of LC11-RNase H1 in complex with an RNA/DNA substrate was determined. Unlike Bacillus halodurans RNase H1 without hybrid binding domain (HBD) (Bh-RNase HC) and human RNase H1 without HBD (Hs-RNase HC), LC11-RNase H1 interacts with four non-consecutive 2'-OH groups of the RNA strand. The lack of interactions with four consecutive 2'-OH groups leads to a dramatic decrease in the ability of LC11-RNase H1 to cleave the DNA-RNA-DNA/DNA substrate containing four ribonucleotides as compared to those to cleave the substrates containing five and six ribonucleotides. The interaction of LC11-RNase H1 with the DNA strand is also different from those of Bh-RNase HC and Hs-RNase HC. Beside the common phosphate-binding pocket, LC11-RNase H1 has a unique DNA-binding channel. Furthermore, the active-site residues of LC11-RNase H1 are located farther away from the scissile phosphate group than those of Bh-RNase HC and Hs-RNase HC. Modeling of Sto-RNase H1 in complex with the 14bp RNA/DNA substrate, together with the structure-based mutational analyses, suggest that the ability of Sto-RNase H1 to cleave double-stranded RNA is dependent on the local conformation of the basic residues located at the DNA binding site.

Accelerated maturation of Tk-subtilisin by a Leu→Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin.

Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin [Tk-subtilisin in a pro form (Gly1-Gly398)], by autoprocessing and degradation of propeptide [Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)]. The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin. To examine whether the mutation of Leu69 to Pro, which is the most unfavourable residue in the P1 position for subtilisins, affects the maturation of Pro-Tk-subtilisin, the Pro-Tk-subtilisin and Tk-propeptide derivatives with this mutation (Pro-L69P and L69P-propeptide) were constructed and characterized. Pro-L69P was autoprocessed more slowly than Pro-Tk-subtilisin. Nevertheless, it matured to Tk-subtilisin more rapidly than Pro-Tk-subtilisin because L69P-propeptide was degraded by Tk-subtilisin more rapidly than Tk-propeptide. The chaperone function and stability of L69P-propeptide were comparable to those of Tk-propeptide, whereas the inhibitory potency and binding ability of L69P-propeptide were considerably reduced compared to those of Tk-propeptide. The crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) revealed that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation. These results suggest that the Leu→Pro mutation accelerates the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin.

Structure and characterization of RNase H3 from Aquifex aeolicus.

The crystal structure of ribonuclease H3 from Aquifex aeolicus (Aae-RNase H3) was determined at 2.0 Å resolution. Aae-RNase H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus stearothermophilus RNase H3 (Bst-RNase H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase H3 is relatively long and flexible, while that of Aae-RNase H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase H3 is important for substrate binding, and uses the flat surface of the ß-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.

Intestinal epithelial cell-derived semaphorin 7A negatively regulates development of colitis via αvß1 integrin.

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Ms) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal Mϕs to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1(+)MHC class II(int)F4/80(hi)CD11b(hi) Mϕs as the main producers of IL-10 via αvß1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by Mϕs during interactions between IECs and Mϕs. Furthermore, we determined that the administration of recombinant Sema7A proteins ameliorated the severity of colitis, and these effects were diminished by IL-10-blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through αvß1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mϕs.

A dual role of divalent metal ions in catalysis and folding of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1.

RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNH1) consists of an N-terminal domain with unknown function and a C-terminal RNase H domain. It is characterized by the high content of acidic residues on the protein surface. The far- and near-UV CD spectra of Halo-RNH1 suggested that Halo-RNH1 assumes a partially folded structure in the absence of salt and divalent metal ions. It requires either salt or divalent metal ions for folding. However, thermal denaturation of Halo-RNH1 analyzed in the presence of salt and/or divalent metal ions by CD spectroscopy suggested that salt and divalent metal ions independently stabilize the protein and thereby facilitate folding. Divalent metal ions stabilize the protein probably by binding mainly to the active site and suppressing negative charge repulsions at this site. Salt stabilizes the protein probably by increasing hydrophobic interactions at the protein core and decreasing negative charge repulsions on the protein surface. Halo-RNH1 exhibited activity in the presence of divalent metal ions regardless of the presence or absence of 3 M NaCl. However, higher concentrations of divalent metal ions are required for activity in the absence of salt to facilitate folding. Thus, divalent metal ions play a dual role in catalysis and folding of Halo-RNH1. Construction of the Halo-RNH1 derivatives lacking an N- or C-terminal domain, followed by biochemical characterizations, indicated that an N-terminal domain is dispensable for stability, activity, folding, and substrate binding of Halo-RNH1.

Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3.

Ribonuclease H3 from Bacillus stearothermophilus (Bst-RNase H3) has the N-terminal TBP-like substrate-binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N-domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate-binding affinities when compared to those of the parent proteins, suggesting that the N-terminal domain of RNase H3 uses the flat surface of the ß-sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding.

The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.

BACKGROUND: The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI). RESULTS: To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins. CONCLUSIONS: These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

Gene cloning and enzymatic properties of hyperthermostable beta-glycosidase from Thermus thermophilus HJ6.

A microorganism (strain HJ6) producing extracellular beta-glycosidase was isolated from a hot springs located in Arima-cho, Hyogo, Japan. The cells were long-rods (2-4 microm) about 0.4 microm in diameter, and formed yellow-colored colonies, like most other strains of the genus Thermus. The pH and temperature for optimal growth were 6.5 and 80 degrees C. Thus, the HJ6 strain displayed a higher optimal temperature than other described Thermus sp. The gene encoding beta-glycosidase (TtbetaGly) was cloned, sequenced, and comprised of 1296 nucleotides encoding a protein (431 amino acids) with a predicted molecular mass of 48.7 kDa. TtbetaGly was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for beta-glycosidase activity were found to be 90 degrees C and 8.5, respectively. The half-life of heat inactivation was about 30 min at 95 degrees C indicating that TtbetaGly had higher thermostability than beta-glycosidases from other Thermus sp. The results of the kinetics experiment indicated that beta-D-fucoside and beta-D-glucoside were better substrates of TtbetaGly than beta-D-galactoside. The catalytic efficiency (k(cat)/K(m)) of TtbetaGly at 80 degrees C increased 70-fold to that at 40 degrees C, indicating that this enzyme was activated at high temperatures. Thin layer chromatography showed that the enzyme had transglycosylation activity at high temperature and that various transfer products were formed in the reaction with lactose or cellobiose.