Background

Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC).

Results

Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis.

Conclusions

Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE).

Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES.

The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C4 substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.

Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5′-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.

Summary

Eukaryotic RNases H2 consist of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi-Goutières syndrome. To examine whether these mutations affect complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding three subunits were co-expressed in E. coli and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced as compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII (Tk-RNase HII). To analyze the role of this residue, four mutant proteins Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P were constructed. All mutant proteins were less stable than the wild-type protein by 2.9–7.6°C in Tm. Comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggest that introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable. The findings that the mutations in the accessory subunits of Sc-RNase H2* do not seriously affect the enzymatic activity suggest that the mutant forms of the protein are relatively unstable or interactions with other proteins are perturbed in human cells.

RNase HI from the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) is stabilized by its C-terminal residues. In this work, the stabilization effect of the Sto-RNase HI C-terminal residues was investigated in detail by thermodynamic measurements of the stability of variants lacking the disulfide bond (C58/145A), or the six C-terminal residues (ΔC6) and by structural analysis of ΔC6. The results showed that the C-terminal does not affect overall structure and stabilization is caused by local interactions of the C-terminal, suggesting that the C-terminal residues could be used as a “stabilization tag.” The Sto-RNase HI C-terminal residues (-IGCIILT) were introduced as a tag on three proteins. Each chimeric protein was more stable than its wild-type protein. These results suggested the possibility of a simple stabilization technique using a stabilization tag such as Sto-RNase HI C-terminal residues.

Adaptation of microorganisms to low temperatures remains to be fully elucidated. It has been previously reported that peptidyl prolyl cis-trans isomerases (PPIases) are involved in cold adaptation of various microorganisms whether they are hyperthermophiles, mesophiles or phsycrophiles. The rate of cis-trans isomerization at low temperatures is much slower than that at higher temperatures and may cause problems in protein folding. However, the mechanisms by which PPIases are involved in cold adaptation remain unclear. Here we used FK506-binding protein 22, a cold shock protein from the psychrophilic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) as a model protein to decipher the involvement of PPIases in cold adaptation. SIB1 FKBP22 is homodimer that assumes a V-shaped structure based on a tertiary model. Each monomer consists of an N-domain responsible for dimerization and a C-catalytic domain. SIB1 FKBP22 is a typical cold-adapted enzyme as indicated by the increase of catalytic efficiency at low temperatures, the downward shift in optimal temperature of activity and the reduction in the conformational stability. SIB1 FKBP22 is considered as foldase and chaperone based on its ability to catalyze refolding of a cis-proline containing protein and bind to a folding intermediate protein, respectively. The foldase and chaperone activites of SIB1 FKBP22 are thought to be important for cold adaptation of Shewanella sp. SIB1. These activities are also employed by other PPIases for being involved in cold adaptation of various microorganisms. Despite other biological roles of PPIases, we proposed that foldase and chaperone activities of PPIases are the main requirement for overcoming the cold-stress problem in microorganisms due to folding of proteins.

The crystal structure of CutA1 from the psychrotrophic bacterium Shewanella sp. SIB1 in a trimeric form was determined at 2.7 Å resolution. This is the first crystal structure of a psychrotrophic CutA1.

CutA1 is widely found in bacteria, plants and animals, including humans. The functions of CutA1, however, have not been well clarified. It is known that CutA1s from Pyrococcus horikoshii, Thermus thermophilus and Oryza sativa unfold at temperatures remarkably higher than the growth temperatures of the host organisms. In this work the crystal structure of CutA1 from the psychrotrophic bacterium Shewanella sp. SIB1 (SIB1–CutA1) in a trimeric form was determined at 2.7 Å resolution. This is the first crystal structure of a psychrotrophic CutA1. The overall structure of SIB1–CutA1 is similar to those of CutA1 from Homo sapiens, Escherichia coli, Pyrococcus horikoshii, Thermus thermophilus, Termotoga maritima, Oryza sativa and Rattus norvergicus. A peculiarity is observed in the β2 strand. The β2 strand is divided into two short β strands, β2a and β2b, in SIB1–CutA1. A thermal denaturation experiment revealed that SIB1–CutA1 does not unfold completely at 363 K at pH 7.0, although Shewanella sp. SIB1 cannot grow at temperatures exceeding 303 K. These results indicate that the trimeric structural motif of CutA1 is the critical factor in its unusually high stability and suggest that CutA1 needs to maintain its high stability in order to function, even in psychrotrophs.

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.

A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed.

A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. The crystal was grown at 277 K by the hanging-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.7 Å resolution using synchrotron radiation at station BL38B1, SPring-8. The crystal belongs to space group P21, with unit-cell parameters a = 48.79, b = 84.06, c = 87.04 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.73 Å3 Da−1 and the solvent content was 55%.

Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed.

Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed. Crystals were grown at 293 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.4 Å resolution using synchrotron radiation at station BL44XU of SPring-8. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 Å. Assuming the presence of two molecules in the asymmetric unit, the V M value was 2.7 Å3 Da−1 and the solvent content was 54.1%. The protein was also cocrystallized with substrates and diffraction data were collected to 2.7 Å resolution.

Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon T. kodakaraensis were performed.

Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.

Type 1 RNase H from the hyperthermophilic archaeon S. tokodaii 7 was overproduced in E. coli, purified, and crystallized. Preliminary crystallographic studies indicated that the crystal belongs to space group P43, with unit-cell parameters a = b = 39.21, c = 91.15 Å.

Crystallization and preliminary crystallographic studies of type 1 RNase H from the hyperthermophilic archaeon Sulfolobus tokodaii 7 were performed. A crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.5 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to space group P43, with unit-cell parameters a = b = 39.21, c = 91.15 Å. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient V M was calculated to be 2.1 Å3 Da−1 and the solvent content was 40.5%. The structure of a selenomethionine Sto-RNase HI mutant obtained using a MAD data set is currently being analysed.

A human kynurenine aminotransferase II homologue from P. horikoshii OT3 has been overproduced in E. coli, purified, and characterized. Crystals of this protein have been obtained and analyzed by X-ray diffraction.

The Pyrococcus horikoshii OT3 genome contains a gene encoding a human kynurenine aminotransferase II (KAT II) homologue, which consists of 428 amino-acid residues and shows an amino-acid sequence identity of 30% to human KAT II. This gene was overexpressed in Escherichia coli and the recombinant protein (Ph-KAT II) was purified. Gel-filtration chromatography showed that Ph-KAT II exists as a homodimer. Ph-KAT II exhibited enzymatic activity that catalyzes the transamination of l-kynurenine to produce kynurenic acid. Crystals of Ph-KAT II were grown using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.2 Å resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the centred orthorhombic space group C2221, with unit-cell parameters a = 71.75, b = 86.84, c = 137.30 Å. Assuming one molecule per asymmetric unit, the V M value was 2.19 Å3 Da−1 and the solvent content was 43.3%.

A thermostable ribonuclease HIII from B. stearothermophilus (Bst RNase HIII) was crystallized and preliminary crystallographic studies were performed. Plate-like overlapping polycrystals were grown by the sitting-drop vapour-diffusion method at 283 K.

A thermostable ribonuclease HIII from Bacillus stearothermophilus (Bst RNase HIII) was crystallized and preliminary crystallographic studies were performed. Plate-like overlapping polycrystals were grown by the sitting-drop vapour-diffusion method at 283 K. Native X-ray diffraction data were collected to 2.8 Å resolution using synchrotron radiation from station BL44XU at SPring-8. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 66.73, b = 108.62, c = 48.29 Å. Assuming one molecule per asymmetric unit, the V M value was 2.59 Å3 Da−1 and the solvent content was 52.2%.

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ∼5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly−82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

We have cloned the gene encoding RNase HII (RNase HIIPk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli. This gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII. RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity was determined at 30°C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E. coli RNase HII, and Mg2+ for E. coli RNase HI. The specific activity of RNase HIIPk determined in the presence of the most preferred metal ion was 6.8-fold higher than that of E. coli RNase HII and 4.5-fold lower than that of E. coli RNase HI. Like E. coli RNase HI, RNase HIIPk and E. coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3′ bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HIIPk and E. coli RNases HI and HII are structurally and functionally related to one another.