Intermediates in guanidine-HC1 unfolding of glutamine synthetase from the extreme thermophile, Bacillus caldolyticus.

Glutamine synthetase is expressed in Bacillus caldolyticus as two isoforms that differ in physico-chemical and regulatory properties. Biphasic kinetics of thermal denaturation of E-I and E-II (Merkler, D.J., et al (1987) Biochemistry 26, 7805), suggested the formation of intermediates. CD spectral changes of E-II induced by guanidine-HC1 clearly indicate a three-state pathway for unfolding (N----I----D). Refolding of E-II from 6 M GuHCl led to only 15% recovery of activity, compared to greater than or equal to 90% with E-I.     

Ribosomal proteins and DNA-binding protein II from the extreme thermophile Bacillus caldolyticus

Crystallographic studies, presently on ribosomal and DNA-binding proteins from the moderate thermophile Bacillus stearothermophilus, can be expected to benefit from the use of even more stable proteins from extreme thermophiles. Bacillus caldolyticus, which is able to grow in the temperature range of 70-80 degrees C, appears to be a suitable candidate. We have compared the two bacilli using two criteria: the two-dimensional gel patterns of ribosomal proteins and the properties of DNA-binding protein II. The latter protein is ubiquitous in the eubacterial kingdom and can be purified in large quantities. B. caldolyticus can be grown at 75 degrees C in continuous culture with a generation time of 45-60 min. The yield of ribosomes compares favorably with that of B. stearothermophilus. The gel patterns of the ribosomal proteins are very similar but several differences, in particular among the 50S proteins, are observed. The N-terminal amino-acid sequence of the DNA-binding protein differs in 3 positions (out of 39) from B. stearothermophilus and the protein shows an increased resistance to thermal denaturation. Tetragonal and monoclinic crystals of DNA-binding protein II have been obtained which are suitable for X-ray studies and the diffraction patterns of the two crystal forms are shown.     

Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8

Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8. As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E. coli and B. stearothermophilus. Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T. thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not. The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another. The conformational transition involving the anticodon of E. coli tRNAGlu as complexed with Glu-tRNA synthetase from T. thermophilus is necessary for the aminoacylation activity.     

Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum.

Thirteen strains of a strict anaerobic, extreme thermophilic bacterium were isolated from soil samples of moderate temperature, from a sewage plant in Georgia, and from hot springs in Utah and Wyoming. They were identified as strains of Clostridium thermohydrosulfuricum. The guanosine + cytosine content (moles percent) was 37.6 (determined by buoyant density) and 34.1 (determined by melting temperature). All strains required a factor present in yeast extract or tryptone growth. Growth characteristics were as follows: a pH range of 5 to 9, with the optimum between 6.9 to 7.5, in a temperature range of 40 to 78 degrees C, with the optimum at 68 degrees C. The doubling time, when grown on glucose at temperature and pH optima, was 1.2 h. The main products of glucose fermentation were ethanol, lactate, acetate, CO2, and H2. The fermentation was inhibited by H2. Formation of spores occurred easily on glucose-agar medium or when cultures growing at temperatures above 65 degrees C were allowed to cool to temperature below 55 degrees C. C. thermohydrosulfuricum occurs widely distributed in the natural environment.     

Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.     

Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM. At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 > KCl = NH4Cl = NaCl > Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.     

Purification and some properties of cytochrome c-552 from an extreme thermophile, Thermus thermophilus HB8.

A c-type cytochrome, cytochrome c-552, from a soluble fraction of an extreme thermophile, Thermus thermophilus HB8, was highly purified and its properties investigated. The absorption peaks were at 552, 522, and 417 nm in the reduced form, and at 408 nm in the oxidized form. The isoelectric point was at PH 10.8, the midpoint redox potential was about +0.23 V, and the molecular weight was about 15,000. The cytochrome c-552 was highly thermoresistant. The cytochrome reacted rapidly with pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2], but slowly with bovine cytochrome oxidase [EC 1.9.3.1], yeast cytochrome c peroxidase [EC 1.11.1.5], or Nitrosomonas europaea hydroxylamine-cytochrome c reductase [EC 1.7.3.4].     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 2. Catalytic properties.

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli. 1. The formation of a ternary guanine-nucleotide . EF-Tu . EF-Ts complex was demonstrated by gel filtration of the T. thermophilus EF-Tu . EF-Ts complex on a Sephadex G-150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli. 2. The dissociation constants for the complexes of T. thermophilus EF-Tu . EF-Ts with GDP and GTP were 6.1 x 10(-7) M and 1.9 x 10(-6) M respectively. On the other hand, T. thermophilus EF-Tu interacted with GDP and GTP with dissociation constants of 1.1 x 10(-9) M and 5.8 x 10(-8) M respectively. This suggests that the association of EF-Ts with EF-Tu lowered the affinity of EF-Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction. 3. Although the T. thermophilus EF-Tu . EF-Ts complex hardly dissociates into EF-Tu and EF-Ts, a rapid exchange was observed between free EF-Ts and the EF-Tu . EF-Ts complex using 3H-labelled EF-Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides. 4. Based on the above findings, an improved reaction mechanism for the regeneration of EF-Tu . GTP from EF-Tu . GDP is proposed. 5. Studies on the functional interchangeability of EF-Tu and EF-Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination. 6. T. thermophilus EF-Ts could bind E. coli EF-Tu to form an EF-Tu (E. coli) . EF-Ts (T. thermophilus hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF-Ts. On the other hand, no stable complex between E. coli EF-Ts and T. thermophilus EF-Tu has been isolated. 7. The uncoupled GTPase activity of T. thermophilus EF-G was much lower than that of E. coli EF-G. T. thermophilus EF-G formed a relatively stable binary EF-G . GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF-G . GDP and EF-G . GTP were 6.7 x 10(-7) M and 1.2 x 10(-5) M respectively. The ternary T. thermophilus EF-G . GDP . ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF-G.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.     

Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile

An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin. No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed. A specificity for small aliphatic amino acids on either side of the splitting point was indicated. Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms. Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h). The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min). None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin. Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days. Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h. At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx. 1 h, 1 h and more than 5 h, respectively.     

Purification and properties of D-glyceraldehyde-3-phosphate dehydrogenase from an extreme thermophile, Thermus thermophilus strain HB 8.

1. D-Glyceraldehyde-3-phosphate dehydrogenase from an extreme thermophile, T. thermophilus strain HB8, was purified and crystallized. 2. The enzyme was found to possess remarkable heat stability, being slowly inactivated at 90 degrees C. 3. Basic kinetic constants and pH profile are reported. The enzyme was activated 25-fold by 90 mM NH4Cl, and also by ethanol up to 5-fold at 30 degrees C. 4. The enzyme was found to be far more resistant to urea or sodium dodecylsulfate than the rabbit enzyme. 5. The enzyme was shown to be a tetramer of molecular weight 130000--135000. Amino acid composition analysis revealed no unusual features. Circular dichroic spectra suggested that the contents of the ordered structure of the thermophile enzyme are similar to those of the rabbit enzyme. 6. The other catalytic properties of the thermophile enzyme are discussed in comparison with those of the enzymes from other sources.     

Cytochrome oxidase from an extreme thermophile. Thermus thermophilus HB8

The cytochrome oxidase (EC 1.9.3.1) of $\text{Thermus}\text{thermophilus}$ HB8 was isolated from the membrane fraction, and was highly purified. The oxidase contained heme $\text{a}$ and heme $\text{c}$ as the prosthetic groups. The purified preparation showed a single band in polyacrylamide gel electrophoresis, and three major polypeptides with apparent molecular weights of 52,000, 37,000 and 29,000 were observed in the presence of sodium dodecyl sulfate. The enzyme reacted rapidly with $\text{T}\text{.}\text{thermophilus}$ cytochrome c-552. The oxidation of $\text{T}\text{.}\text{thermophilus}$ cytochrome $\text{c}$-555,549 by the enzyme was very slow, and was stimulated by the addition of cytochrome $\text{c}$-552. The enzyme was highly stable to heat.     

Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25 °C for enzyme reaction.     

A new polyamine, thermospermine, 1,12-diamino-4,8-diazadodecane, from an extreme thermophile.

A new polyamine has been extracted from an extreme thermophile, Thermus thermophilus, and its chemical structure was determined as 1,12-diamino-4,8-diazadodecane, NH2(CH2)3NH(CH2)3NH(CH2)4NH2, based on its proton NMR, 13C NMR, and mass spectra. A trivial name "thermospermine" is proposed for the new compound.