Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus aquaticus YT-1 and the deduced amino acid sequence of the enzyme

The gene for L-lactate dehydrogenase (LDH) from Thermus aquaticus YT-1 was cloned in Escherichia coli, using the Thermus caldophilus LDH gene as a hybridization probe, and its complete nucleotide sequence was determined. The LDH gene comprised 930 base pairs, starting with a GTG initiation codon. Its sequence had high homology (85.8% identity) with the LDH gene of T. caldophilus. The G + C content of the T. aquaticus gene was 70.9%, higher than that of the chromosomal DNA (67.4%). In particular, that in the third position of the codons used was 91.0%, similar to the T. caldophilus gene. The primary structure of T. aquaticus LDH was deduced from the nucleotide sequence of the LDH gene. It comprises 310 amino acid residues, as does T. caldophilus LDH, and its molecular mass was calculated to be 33,210 daltons. The amino acid sequence of the T. aquaticus LDH had 87.1% identity with that of the T. caldophilus LDH. At 23 positions, the respective residues differed in charge and polarity. These differences must be related to the differences in kinetic properties between the two enzymes. The constructed plasmid overproduced the T. aquaticus LDH in E. coli.     

Fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase from Thermus aquaticus YT-1, an extreme thermophile: activation by citrate and modification reagents and comparison with Thermus caldophilus GK24 L-lactate dehydrogenase

Heat-stable fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase [EC 1.1.1.27] was purified from an extremely thermophilic bacterium, Thermus aquaticus YT-1. The amino acid composition and NH2-terminal 34 amino acid sequence of the enzyme were determined. Its NH2-terminal sequence shows high homology with those of Thermus caldophilus GK24 (82% identity) and some other bacterial L-lactate dehydrogenases (44-53% identity), indicating the close phylogenic relationship of the two Thermus species. At the same time, the two Thermus L-lactate dehydrogenases were found not to be identical not only chemically but also kinetically and immunologically. Citrate activated the T. aquaticus enzyme in the weak acidic pH region, while fructose 1,6-bisphosphate did in both acidic and neutral pH regions. The maximum activity obtained with citrate at pH 5.0 was about 2.5 times higher than that in the presence of fructose 1,6-bisphosphate at pH 6.7. The enzymes modified with 2,3-butanedione, acetic anhydride and diethyl pyrocarbonate in the presence of both NADH and oxamate were desensitized to fructose 1,6-bisphosphate, and the modified enzymes were active even in the absence of fructose 1,6-bisphosphate. All of the modified enzymes examined were still activated by citrate similarly to the native enzyme. These results suggest that the mechanism of activation by citrate is different from that by fructose 1,6-bisphosphate, and that the citrate-binding site is different from the fructose 1,6-bisphosphate-binding site.     

Allosteric effect of fructose 1,6-bisphosphate on the conformation of NAD+ as bound to L-lactate dehydrogenase from Thermus caldophilus GK24

The allosteric effect of fructose 1,6-bisphosphate (Fru-1,6-P2) on L-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from Thermus caldophilus GK24 was studied by means of 1H NMR analyses. The conformation of NAD+ as bound to the T. caldophilus enzyme was elucidated by analyses of the transferred nuclear Overhauser effects (TRNOE), in the presence and the absence of the allosteric effector, Fru-1,6-P2. Upon binding of Fru-1,6-P2 to the enzyme, the ribose ring of the adenosine moiety of NAD+ is converted from the C2'-endo form to the C3'-endo form. This C3'-endo form of the adenosine moiety is similar to that of NAD+ as bound to nonallosteric vertebrate enzymes. However, the anti conformation of the adenine-ribose bond of NAD+ as bound to the T. caldophilus enzyme is not affected by the binding of Fru-1,6-P2. In contrast, the syn conformation of the nicotinamide-ribose bond is converted to the anti form on the binding of Fru-1,6-P2, while the ribose ring remains in the C3'-endo form as found in the case of a nonallosteric enzyme. Such a conformational change of enzyme-bound NAD+ as found on TRNOE analysis is essentially involved in the allosteric regulation of the T. caldophilus enzyme by Fru-1,6-P2.     

Involvement of the conserved histidine-188 residue in the L-lactate dehydrogenase from Thermus caldophilus GK24 in allosteric regulation by fructose 1,6-bisphosphate

The conserved histidine-188 residue of the L-lactate dehydrogenase of Thermus caldophilus GK 24, which is allosterically activated by fructose 1,6-bisphosphate, has been exchanged to phenylalanine by site-specific mutagenesis. In the mutant enzyme the strong stimulatory effect of fructose 1,6-bisphosphate is abolished. The analysis of the pH dependence of the activity indicates that the positive charge of the conserved His-188 residue is important for the interaction of the enzyme with the allosteric effector.     

Crystallization of allosteric L-lactate dehydrogenase from Thermus caldophilus and preliminary crystallographic data

L-Lactate dehydrogenase of Thermus caldophilus GK24 was purified from Escherichia coli containing an overexpression plasmid. The enzyme was crystallized from polyethylene glycol 6000 solutions without ligands by the hanging drop vapor diffusion method. Two forms of crystals were obtained. The crystals grown at pH 6.0 were characterized by means of an X-ray diffraction experiment, while those grown at pH 6.5 and 7.0 did not give detectable diffraction spots. The crystals grown at pH 6.0 belonged to monoclinic space group P2(1), the cell dimensions being a = 54.8 A, b = 138.2A, c = 86.1 A, and beta = 93.3 degrees. These crystals diffract to beyond 2.5 A spacing and are stable on X-ray irradiation.     

Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium

Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).     

Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.     

Nucleotide sequence of the Saccharomyces cerevisiae CTT1 gene and deduced amino-acid sequence of yeast catalase T

A 2642-base-pair DNA fragment containing the catalase T (CTT1) structural gene of the yeast Saccharomyces cerevisiae and its flanking regions has been sequenced. The gene codes for a protein of 562 amino acids (relative molecular mass 64,449) and appears to contain no intron. The amino acid sequence of catalase T derived from the DNA sequence shows 40.7% homology (52.2% including conservative replacements) to that of bovine liver catalase. All amino acids previously postulated to participate directly in catalysis by liver catalase and most of the amino acids of the immediate environment of hemin, the prosthetic group of catalase, are conserved in catalase T. The data obtained indicate that the folding of polypeptide chains of the two catalases compared has been conserved within a central region consisting mainly of the beta-barrel domain, which bears the prosthetic group, and a major part of the "wrapping domain". N- and C-terminal regions involved in subunit interactions are less well conserved. It is suggested that their structure is more similar to that of the corresponding regions of Penicillium vitale catalase. However, catalase T lacks the C-terminal flavodoxin-like domain present in this protein.     

Nucleotide sequence of the full-length mouse lamin C cDNA and its deduced amino-acid sequence

We have cloned and sequenced the cDNA comprizing the entire coding region and several hundred base-pairs of its flanks for the mouse nuclear envelope protein lamin C mRNA. The nucleotide sequence and the deduced amino-acid sequence of the mouse lamin C are compared with the previously published human lamin A/C sequences with respect to (a) the general organisation, (b) homologies, (c) predictions for the essential structural characteristics of lamins and (d) the localization of the most conserved region. Moreover, the mouse lamin C sequence presented allows the first intraspecies comparison between A/C-type and B-type lamins.     

Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase

This paper questions what types of molecular transformation are involved in the conversion of maltose to trehalose by trehalose synthase from Thermus caldophilus GK24. The reverse reaction pathway has been examined with the aid of alpha,alpha-(2,4,6,6',2',4',6",6"'-(2)H(8))trehalose (1). The mass data of the isolated reaction products clearly indicate that deuterated glucose is confined only to substrate molecules, and thus the reversible enzymatic conversion of trehalose into maltose proceeds through an intramolecular pathway.     

Nucleotide sequence of the malate dehydrogenase gene of Thermus flavus and its mutation directing an increase in enzyme activity

The nucleotide sequence of the malate dehydrogenase (mdh) gene from a thermophilic bacterium, Thermus flavus, was determined. The amino acid sequence of the Thermus malate dehydrogenase resembled that of the porcine heart cytoplasmic enzyme to a certain extent, and Asp-159 and His-187 were identified as possible essential residues for the catalytic function. The mutated mdh gene was also cloned from a spontaneous mutant of T. flavus containing a higher activity of the enzyme. Its mutation point was determined to be a single nucleotide exchange from C to T which caused Thr-190 to be substituted by isoleucine. The mutated enzyme showed resistance to substrate inhibition, an increase in both kcat and Km, and a shift toward a more acid optimum pH for the enzyme reaction.     

Cloning, expression, and biochemical characterization of a new histone deacetylase-like protein from Thermus caldophilus GK24

Histone deactylases (HDACs) are members of an ancient enzyme family found in eukaryotes as well as in prokaryotes such as archaebacteria and eubacteria. We here report a new histone deacetylase (Tca HDAC) that was cloned from the genomic library of Thermus caldophilus GK24 based on homology analysis with human histone deacetylase1 (HDAC1). The gene contains an open reading frame encoding 375 amino acids with a calculated molecular mass of 42,188 Da and the deduced amino acid sequence of Tca HDAC showed a 31% homology to human HDAC1. The Tca HDAC gene was over-expressed in Escherichia coli using a Glutathione-S transferase (GST) fusion vector (pGEX-4T-1) and the purified protein showed a deacetylase activity toward the fluorogenic substrate for HDAC. Moreover, the enzyme activity was inhibited by trichostatin A, a specific HDAC inhibitor, in a dose-dependent manner. Optimum temperature and pH of the enzyme was found to be approximately 70 degrees C and 7.0, respectively. In addition, zinc ion is required for catalytic activity of the enzyme. Together, these data demonstrate that Tca HDAC is a new histone deacetylase-like enzyme from T. caldophilus GK24 and will be a useful tool for deciphering the role of HDAC in the prokaryote and development of new biochemical reactions.     

Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25-28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2-terminal sequence previously reported and the determined amino acid sequences, including the COOH-terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28,350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin-type serine proteases, and 43% identity with proteinase K, 37-39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2-terminal portion, the protease and a COOH-terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68-74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.     

L-Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium. Desensitization to fructose 1,6-bisphosphate in the activated state by arginine-specific chemical modification and the N-terminal amino acid sequence

Heat-stable and fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91, 1343-1348]. N-terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N-terminal arm region (first 1-20 residues) known for the vertebrate L-lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD-binding domain. The C-terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate L-lactate dehydrogenases. On modification reaction with 2,3-butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilus L-lactate dehydrogenase was obtained independently of fructose 1,6-bisphosphate, and the modified enzyme was desensitized to fructose 1,6-bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6-bisphosphate. A similar desensitization was observed using 1,2-cyclohexanedione instead of 2,3-butanedione. The enzyme was irreversibly modified with 2,3-butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6-bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6-bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

Abstract The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EII^Man) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EII^Man showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EII^Glc). Similar homology was shown between the C-terminal sequence of EII^Man and the E. coli glucose-specific enzyme III (EIII^Glc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EII^Man contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIII^Glc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.