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At least 253 records · Page 14Linked to original sources
PubMed1993

The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent.

The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 A, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 A for both bonds and planes. The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53 degrees C, respectively. Differential scanning calorimetry indicates that the energy difference (delta delta G) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol-1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better alpha-helix stability resulting from the removal of helix destabilizing residues and extra helix-dipole/helix side chain ionic interactions.

Enzyme Stability↗
PubMed1994

Comparison of the inhibition by phospho(enol)pyruvate and phosphoglycolate of phosphofructokinase from B. stearothermophilus.

A comparison between the inhibition by phospho(enol)pyruvate (PEP) versus the inhibition by phosphoglycolate (PG) of phosphofructokinase (PFK) from Bacillus stearothermophilus is presented. Both inhibitors act by decreasing the apparent affinity displayed by the enzyme for its substrate fructose 6-phosphate (Fru-6-P) while having little effect on Vmax. However, the two ligands differ in both their affinity for the enzyme and their effectiveness at antagonizing the subsequent binding of Fru-6-P. Although PG binds with approximately 10-fold lower affinity, it antagonizes the binding of Fru-6-P 3.5-fold more strongly than does PEP. Moreover, the enthalpy and entropy contributions to the coupling free energy between inhibitor and Fru-6-P, from which these antagonisms derive, reveal even greater differences between the ligands. These data indicate, therefore, that the changes in the structure of PFK from B. stearothermophilus that result from PG binding, which have been determined by X-ray crystallography (T. Schirmer and P. R. Evans, 1990 Nature 343, 140-145), may not be comparable to those that result from PEP binding and consequently do not represent the generic "T-state," as has been presumed.

Calorimetry↗
PubMed1997

Characterization of metal and nucleotide liganded forms of adenylate kinase by electrospray ionization mass spectrometry.

Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis, and Bacillus stearothermophilus with the bisubstrate nucleotide analog P1,P5-di(adenosine 5')-pentaphosphate and with metal ions (Zn2+ and/or Mg2+) were analyzed by electrospray ionization mass spectrometry. P1,P5-di(adenosine 5')-pentaphosphate. adenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleotide to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2+ chelated form of the enzyme from the gram-positive organisms. In this way, it is possible in a single mass spectrometry experiment to screen metal-chelating adenylate kinases, without use of radioactively labeled compounds. Binding of Mg2+ to enzyme via P1,P5-di(adenosine 5')-pentaphosphate was also demonstrated by mass spectrometry. Although no amino acid side chain in adenylate kinase is supposed to interact with Mg2+, Asp93 in porcine muscle cytosolic enzyme, equivalent to Asp84 in the E. coli adenylate kinase, was proposed to stabilize the nucleotide.Mg2+ complex via water molecules.

Adenylate Kinase↗
PubMed1997

The N-terminal sequence of Lactococcus lactis phosphoglucose isomerase purified by affinity chromatography differs from the other species.

A specific monoclonal antibody, M3A, was produced to rapidly purify Lactococcus lactis phosphoglucose isomerase (PGI) for amino acid sequence analysis. M3A recognized the Lac. lactis PGI specifically and sensitively with both enzyme-linked immunosorbent assay and Western blot analysis. The enzyme was rapidly purified to a specific activity of 21.8 U/mg with a yield of 20% by a three-step procedure, including M3A-bound Sepharose chromatography. The specific activity of PGI was increased about 64.1-fold from the cell lysate. The molecular mass of Lac. lactis PGI was estimated to be about 50 kDa by SDS-PAGE. The N-terminal amino acid sequence of Lac. lactis PGI exhibited no significant similarity to other PGIs, except for a 52.6% identity to Bacillus stearothermophilus PGI A and PGI B. These results suggest that there might be some molecular types of PGI.

Amino Acid Sequence↗
PubMed1999

The active site of phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not designed to increase the nucleophilicity of a serine residue.

Changing a catalytic cysteine into a serine, and vice versa, generally leads to a dramatic decrease in enzymatic efficiency. Except a study done on thiol subtilisin, no extensive study was carried out for determining whether the decrease in activity is due to a low nucleophilicity of the introduced amino acid. In the present study, Cys149 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus was converted into a Ser residue. This leads to a drastic reduction of the kcat value. The rate-limiting step occurs before the hydride transfer step. Selective, but slow, inactivation is observed with specific, structurally different, inhibitors of serine protease. The esterolytic activity of serine mutant towards activated esters is also strongly decreased. The rate-limiting step of the esterase reaction also shifts from deacylation in the wild type to acylation in the mutant. Altogether, these results strongly suggest that the low catalytic efficiency of the Ser mutant is due to a poor nucleophilicity of the hydroxyl serine group within the active site of the enzyme. The fact that (1) the apo --> holo transition does not change esterolytic and inactivating efficiencies, and (2) Ser149 Asn176 double mutant exhibits the same chemical reactivity and esterolytic catalytic efficiency compared to the Ser149 single mutant indicates that the serine residue is not subject to His176 general base catalysis. A linear relationship between the catalytic dehydrogenase rate, the kcat/KM for esterolysis, and the concentration of OH- is observed, thus supporting the alcoholate entity as the attacking reactive species. Collectively this study shows that the active site environment of GAPDH is not adapted to increase the nucleophilicity of a serine residue. This is discussed in relation to what is known about Ser and Cys protease active sites.

Amino Acid Substitution↗
PubMed1997

A colorimetric assay for phosphate to measure amplicon accumulation in polymerase chain reaction.

We describe a rapid colorimetric method for the detection of PCR products. Color generation is complete within 5 min of mixing reagents with a PCR. The method is simple and does not require affinity capture steps or special labeling to be carried out. The color development can be monitored by eye or a simple spectrophotometer can be used to read sample absorbance. We demonstrate the method by its use in the amplification refractory mutation system (ARMS) analysis [C. R. Newton, A. Graham, L. E. Heptinstall, S. J. Powell, C. Summers, N. Kalsheker, et al. (1989) Nucleic Acids Res. 17, 2503-2515] of cystic fibrosis [J. R. Riordan, J. M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, et al. (1989) Science 245, 1066-1073] and factor V [R. M. Bertina, B. P. C. Koeleman, T. Koster, F. R. Rosendaal, R. J. Dirven, H. de Ronde, P. A. van der Velden, and P. H. Reitsma (1994) Nature 369, 64-67] allelic variants.

Alleles↗
PubMed2001

Mass spectrometry as a novel approach to probe cooperativity in multimeric enzymatic systems.

Investigating cooperativity in multimeric enzymes is of utmost interest to improve our understanding of the mechanism of enzymatic regulation. In the present article, we propose a novel approach based on mass spectrometry to probe cooperativity in the binding of a ligand to a multisubunit enzyme. This approach presents the selective advantage of giving a direct insight into all the subsequent ligation states that are formed in solution as the ligand is added to the enzyme. A quantitative interpretation of the electrospray ionization (ESI) mass spectra gives the relative abundance of all the distinct enzymatic species, which allows one to directly deduce the cooperativity of the system. The overall method is described for the addition of the oxidized cofactor nicotinamide adenine dinucleotide (NAD(+)) to a dimeric mutant of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase (GPDH). It is then applied to four tetrameric enzymes: sturgeon muscle GPDH, wild type and S48G mutant of GPDH from B. stearothermophilus, and alcohol dehydrogenase (ADH) from Bakers yeast. The results illustrate the possibilities offered by this new technique. First, mass spectrometry allows a control of the enzymes before the addition of NAD(+). Second, the cooperative behavior can be drawn from one single ESI mass spectrum, which makes the method highly attractive in terms of the amount of biological material required. Above all, the major benefit lies in the direct visualization of all the enzymatic species that are in equilibrium in solution. The direct measurement of cooperativity readily resolve the inconvenience of the classical approaches employed in this field, which all need to model the experimental data in order to get the cooperative behavior of the system.

Animals↗
PubMed2001

Introduction of a (poly)histidine tag in L-lactate dehydrogenase produces a mixture of active and inactive molecules.

A (poly)histidine tag was fused to either the N- or the C-terminus of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5'-AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein.

Chromatography, Affinity↗
PubMed2002

A novel fluorescence competitive assay for glucose determinations by using a thermostable glucokinase from the thermophilic microorganism Bacillus stearothermophilus.

We describe the use of a thermostable glucokinase in a novel competitive fluorescence assay for glucose. Glucokinase from Bacillus stearothermophilus (BSGK) was found to retain enzymatic activity in solution for over 20 days. The single cysteine residue in BSGK, which is near the active site, was labeled with a fluorescent probe, 2-(4-iodoacetamidoanilino)naphthalene-6-sulfonic acid. The ANS-labeled BSGK displayed a modest 25% decrease in the emission intensity upon binding glucose but no change in lifetime. To obtain a larger spectral change we developed a competitive assay for glucose using the intrinsic tryptophan fluorescence from BSGK and a resonance energy transfer (RET) acceptor-labeled sugar. The sugar-labeled acceptor quenched the BSGK tryptophan emission, and the quenching was reversed upon addition of glucose. The use of RET as the sensing mechanism can be easily extended to longer wavelengths for a more practical glucose sensor.

Enzyme Stability↗
PubMed1993

Similarities between alanine dehydrogenase and the N-terminal part of pyridine nucleotide transhydrogenase and their possible implication in the virulence mechanism of Mycobacterium tuberculosis.

Recent developments in simultaneous multiple alignment methods of protein sequences allow prediction of structural similarity in related proteins. Alanine dehydrogenase and the N-terminal sequence of pyridine nucleotide transhydrogenase were compared for their sequences. High similarities of sequences were observed especially in their NAD(H)-binding sites. These similarities suggest that antibodies which recognized the alanine dehydrogenase of Mycobacterium tuberculosis can also be directed against the membrane bound pyridine nucleotide transhydrogenase. If this is the case, the virulent property of this pathogen could be linked to its higher synthesis of NADPH necessary for its anabolism.

Alanine Dehydrogenase↗
PubMed1993

The primary structure of phosphofructokinase from Lactococcus lactis.

The primary amino acid sequence of phosphofructokinase (EC2.7.1.11) from Lactococcus lactis, obtained by Edman analysis of peptides obtained from proteolytic digestions, is MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGIEVYGINHGYAGMVAGDIF PLTSASVGDKIGRGGTFLYSARYPEFAQVEGQLAGIEQLKKFGIEGVVVI GGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDFTIGFDTAVSTVVDAL DKIRDTSSSHNRTFVVEVMGRNAGDIALNAGIAAGADDISIPELEFKFEN VVNNINKGYEKGKNHHIIIVAEGVMTGEEFATKLKEAGYKGDLRVSVLGH IQRGGSPTARDRVLASRMGARAVELLRDGIGGVAVGIRNEELVESPILGT AEEGALFSLTTEGGIKVNNPHKAGLELYRLNSALNNLNL.

Amino Acid Sequence↗
PubMed1995

Improvement of thermal stability of subtilisin J by changing the primary autolysis site.

The thermostability of subtilisin J, an extracellular serine protease secreted from Bacillus stearothermophilus, has been improved by changing the primary autolysis site of the Asp-49 mutant protein. Previously we have shown that the Asp-49 mutant protein has proteolytic activity, but so unstable that it was primarily autolyzed in Tyr-58-Gln-59 peptide bond during cultivation (Jang et al. Biochim. Biophys. Acta. 1162, 233-235 1993). In the present study, to mitigate the autolytic degradation and increase the thermostability, we deleted the Tyr-58 residue using the Asp-49 mutant as a template. This mutant (Asp-49/delta Tyr-58 mutant) protein showed an improved resistance to heat treatment without changing the catalytic efficiency of the enzyme. These results show that change of primary autolysis site can stabilize the subtilisin.

Amino Acid Sequence↗
PubMed1995

Significance of Phe-220 and Gln-221 in the catalytic mechanism of farnesyl diphosphate synthase of Bacillus stearothermophilus.

Farnesyl diphosphate synthase [EC 2.5.1.10] from Bacillus stearothermophilus was specifically altered at two amino acid residues by using site-directed mutagenesis. The highly conserved Phe and Gln residues at the sequential amino acid positions 220-221 in an upstream part of the putative substrate binding site were replaced with Ala and Glu, respectively. These mutageneses (F220A and Q221E) resulted in 10(-5) and 10(-3) decreases in catalytic activity of farnesyl diphosphate synthesis, respectively. Michaelis constants of the Q221E mutant for the allylic substrates (dimethylallyl- and geranyl diphosphates) increased approximately 25- and 2-folds, respectively, compared to wild type, whereas those for the homoallylic substrate (isopentenyl diphosphate) were not altered much. These results suggest that the Phe-Gln motif is involved not only in the binding of allylic substrates but also in the catalysis by farnesyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed1998

C-terminal regions of D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure.

Most microbial D-hydantoinases have been reported to have catalytic properties similar to those of mammalian dihydropyrimidinases. Comparison of the primary structures of microbial D-hydantoinases with mammalian dihydropyrimidinases revealed that the amino acid homology is about 37% and functionally important residues are rigidly conserved at identical positions. Interestingly, however, the C-terminal regions were found to be completely mismatched with each other. In order to investigate the possible role of the C-terminal regions, we deleted the C-terminal regions of the D-hydantoinases from two thermophilic Bacilli and compared the catalytic and structural properties of the mutant enzymes with those of wild-type enzymes. As a result, the C-terminal region was found not to be essential for catalysis, but it does affect the oligomeric structure of the enzyme.

Amidohydrolases↗
PubMed2001

Fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus: its construction and reconstitution of active fragmentary enzyme.

X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)(+)-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693-705). To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD(+)-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of l-leucine, l-isoleucine, and l-valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50 degrees C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site.

Amino Acid Oxidoreductases↗
PubMed2001

Pressure denaturation of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The effects of hydrostatic pressure on apo wild-type glyceraldehyde-3-phosphate dehydrogenase (wtGAPDH) from Bacillus stearothermophilus (B. stearothermophilus) have been studied by fluorescence spectroscopy under pressure from 0.1 to 650 MPa. Unlike yeast GAPDH [Ruan, K. C., and Weber, G. (1989) Biochemistry 28, 2144-2153], denaturation of the tetrameric apo wtGAPDH from B. stearothermophilus is likely to precede dissociation into subunits. As expected, denaturation is accompanied by the loss of enzymatic activity. B. stearothermophilus apo wtGAPDH interfaces are less pressure sensitive than apo yeast GAPDH ones, while NAD does not protect B. stearothermophilus wtGAPDH against denaturation by pressure. The pressure effects on B. stearothermophilus GAPDH whose R and Q-axis interfaces were destabilized by disruption of interfacial hydrogen bonds are similar to that of apo wtGAPDH.

Apoenzymes↗
PubMed2001

Structural modeling and characterization of a thermostable lipase from Bacillus stearothermophilus P1.

The moderate thermophilic bacterium Bacillus stearothermophilus P1 expresses a thermostable lipase that was active and stable at the high temperature. Based on secondary structure predictions and secondary structure-driven multiple sequence alignment with the homologous lipases of known three-dimensional (3-D) structure, we constructed the 3-D structure model of this enzyme and the model reveals the topological organization of the fold, corroborating our predictions. We hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and identified Ser-113, Asp-317, and His-358 as the putative members of the catalytic triad that are located close to each other at hydrogen bond distances. In addition, the strongly inhibited enzyme by 10 mM PMSF and 1-hexadecanesulfonyl chloride was indicated that it contains a serine residue which plays a key role in the catalytic mechanism. It was also confirmed by site-directed mutagenesis that mutated Ser-113, Asp-317, and His-358 to Ala and the activity of the mutant enzyme was drastically reduced.

Amino Acid Sequence↗
PubMed2001

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.

Amino Acid Sequence↗