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At least 595 records · Page 33Linked to original sources
PubMed1992

Role of residue Glu152 in the discrimination between transfer RNAs by tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

Residue Glu152 of tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is close to phosphate groups 73 and 74 of tRNATyr in the structural model of their complex. TyrTS(E152A), a mutant synthetase carrying the change of Glu152 to Ala, was toxic when overproduced in Escherichia coli. The toxicity strongly increased with the growth temperature. It was measured by the ratios of the efficiencies with which the producing cells plated in induced or repressed conditions and at 30 degrees C or 37 degrees C. TyrTS(E152Q), TyrTS(E152D) and the wild-type synthetase were not toxic in conditions where TyrTS(E152A) was toxic. The toxicity of TyrTS(E152A) was abolished by additional mutations of the synthetase that prevent the binding of tRNATyr but not by a mutation that prevents the formation of Tyr-AMP. Because TyrTS(E152A) was active for the aminoacylation of tRNATyr, its toxicity could only be due to faulty interactions with non-cognate tRNAs, either their non-productive binding or their mischarging with tyrosine. TyrTS(E152A) and TyrTS(E152Q) mischarged tRNAPhe and tRNAVal in vitro with tyrosine unlike TyrTS(E152D) or the wild-type enzyme. Thus, several features of the side-chain in position 152 of TyrTS, including its negative charge, are important for the rejection of non-cognate tRNAs. TyrTS(E152A), TyrTS(E152D) and TyrTS(E152Q) had similar steady-state kinetics parameters for the charging of tRNATyr with tyrosine in vitro, with kcat/KM ratios improved 2.5 times relative to the wild-type synthetase. We conclude that the side-chain of residue Glu152 weakens the binding of TyrTS to tRNATyr and prevents its interaction with non-cognate tRNAs.

Adenosine Monophosphate↗
PubMed1994

Molecular recognition in proteins. Simulation analysis of substrate binding by a tyrosyl-tRNA synthetase mutant.

Alchemical molecular dynamics simulations are performed to determine the difference in the free energy of binding of the tyrosine substrate between the wild type of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and the mutant Tyr169-->Phe. The results are of general interest because the Tyr169 hydroxyl group interacts with the ammonium group of the substrate in a manner corresponding to that found in other amino acid binding proteins (e.g. the Asp receptor of the chemotactic bacterium Salmonella typhimurium and class I major histocompatibility complex molecules). The calculated free-energy change due to the Tyr169-->Phe mutation is 3.4 kcal/mol (the statistical error is +/- 0.5 kcal/mol) in satisfactory agreement with the experimental value of 3(+/- 0.5) kcal/mol. By use of thermodynamic integration, the contribution of the different terms to the free energy change are estimated. The path dependence of such a decomposition is discussed and it is suggested that the alchemical choice is of primary interest for understanding the interactions involved. There are large protein contributions to the alchemical free energy difference of the bound and free enzyme that cancel in the overall result. Due to this cancellation, the essential interactions contributing to the free-energy change are those between the OH group of Tyr169 and water in the free enzyme and those between the OH group of Tyr169 and the ammonium group of the substrate in the bound system. The results thus support simple models based on a balance of hydrogen bonding interactions.

Bacterial Proteins↗
PubMed1983

Specific ligand-induced association of an enzyme. A new model of dissociating allosteric enzyme.

The kinetic behavior of dissociative enzyme system of the type inactive monomer in equilibrium active dimer where dimeric form is stabilized by specific ligand (in particular by substrate) which is bound in the region of the contact of monomers has been analysed. It is assumed that the dissociation of dimer results in formation of monomers which retain the subsites for specific ligand binding. The shape of the dependences of enzyme reaction rate (v) on substrate concentration (S) has been characterized using the order of enzyme reaction rate with respect to substrate concentration: ns = d ln v/d ln [S]. When the substrate concentrations are low the dependences of v on [S] have S-shaped form (the maximum value of ns exceeds the unity) at the definite values of the parameters of the enzyme system. The value of ns approaches--2 at sufficiently high substrate concentrations (in the region where the substrate reveals the inhibitory effect due to blocking the association of inactive monomers into active dimer). The methods of calculation of the parameters of the dissociative enzyme system under discussion have been elaborated on the basis of the analysis of the experimental dependences of specific enzyme activity on enzyme concentration obtained at various fixed substrate concentrations.

Allosteric Regulation↗
PubMed1980

Use of the carbon dioxide laser in sterilization of endodontic reamers.

The object of this study was to test a new method of sterilizing endodontic instruments by using a carbon dioxide laser system. This was done by contaminating endodontic reamers with spores, exposing them to a CO2 laser beam, and checking for their viability by standard microbiologic techniques. It was found that 100 percent of the spores were killed by the CO2 laser. This holds promise as an effective method of sterilizing endodontic instruments in the future.

Bacillus subtilis↗
PubMed1982

Ultrasonic potentiation of the sporicidal activity of glutaraldehyde.

Laboratory test data showed a 4.8-fold increase in the sporicidal activity of glutaraldehyde with the incorporation of ultrasound. Projected times required for 100 percent killed of a 20,000 hydrated spore inoculant were reduced from 211 minutes to 44 minutes. Clinical exposure time intervals necessitated a 31/2-hour glutaraldehyde immersion versus a 30-minute cavitated glutaraldehyde chemosterilization to ensure a 100 percent kill. The feasibility of reducing the ADA-required 10-hour soak in glutaraldehyde to ensure sterility with the incorporation of ultrasonic baths is supported by these data. Tests performed also showed that the airborne contamination from an open tank method is minimal. No spores were retrieved from vapor collected by passive vacuum during cavitation of inoculated glutaraldehyde.

Aldehydes↗
PubMed1984

The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus).

In a previous study, a mutant of tyrosyl-tRNA synthetase in which a threonine residue (Thr51) was converted to proline dramatically improved the affinity of the enzyme for its ATP substrate. How does Pro51 improve the enzyme's affinity for ATP? A priori, Pro51 might interact directly with the ATP, or it might distort the polypeptide backbone and thereby force new or improved contacts elsewhere from the enzyme to ATP. By making mutants of the Pro51 enzyme at two residues that make hydrogen bonds to the ATP substrate, we show that Pro51 greatly improves the strength of one of these contacts. Thus the propagation of a structural change in an enzyme induced by mutation may be detected by the introduction of further mutations.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Electrochemical bioreactor with immobilized glucose-6-phosphate dehydrogenase on the rotating graphite disc electrode modified with phenazine methosulfate.

Physical adsorption, covalent binding through the carbodiimide reaction between the surface carboxyl group and the amino group of the protein, and the crosslinking method with bovine serum albumin by glutaraldehyde were applied for the immobilization of glucose-6-phosphate dehydrogenase (G6PDH) on a graphite electrode. Among those, the crosslinking method was employed for its highest apparent enzyme activity per unit surface area. Phenazine methosulfate (PMS), as a mediator for the electrochemical oxidation of NADH, was also immobilized on the graphite surface through adsorption. The conjugation reaction of G6PDH and the electrochemical oxidation of NADH were confirmed by cyclic voltammetry and the constant potential electrochemical reaction. An electrochemical bioreactor system was established by using a rotating disc graphite electrode with G6PDH immobilized. The coenzyme, NAD, was effectively recycled between the electrochemical and the enzymatic reactions.

Biotechnology↗
PubMed1993

Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents.

A series of variants of kanamycin nucleotidyltransferase (KNTase), isolated previously on the basis of enhanced thermostability by cloning and selection for enzymatic activity in the thermophile Bacillus stearothermophilus, was used to systematically test the hypothesis that thermostable enzymes would also be more resistant to other forms of protein denaturation. The purified KNTases were treated with proteinase K or assayed at 37 degrees C in the presence of urea, N-lauroylsarcosine, Triton X-100, tetrahydrofuran, ethanol, or dimethylformamide. With all these agents, the KNTases displayed increasing resistance to denaturation in the order: wild type, mutant TK9 (with a Thr130-->Lys substitution), TK1 (Asp80-->Tyr), and TK101 (both substitutions). This is the same order in which their thermostability increases, indicating that the structural mechanism(s) whereby the mutations yield enhanced resistance to heat denaturation also yield stabilization towards chemical forms of enzymatic inactivation. These results suggest that selection in thermophiles is a useful method to obtain enzyme variants with increased overall stability, even at nonthermophilic temperatures.

Detergents↗
PubMed1995

Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity.

An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery. The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C. The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration. Native polyacrylamide gels stained for esterase activity showed three bands. The isoelectric points were estimated to be 5.7, 5.8, and 6.0. Forty amino acid residues were sequenced at the N-terminus. The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0. At this pH and 60 degrees C the half-life was 170 h. Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid. The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent. The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths).

Amino Acid Sequence↗
PubMed1993

Does the increased hydrophobicity of the interior and hydrophilicity of the exterior of an enzyme structure reflect its increased thermostability?

The values of hydrophobicity of internal and external elements of the secondary structure of three Bacillus alpha-amylase (beta alpha)8 barrel domains have been calculated in order to investigate whether there is some correlation between the values and the enzyme stability. All the values have been referred to the number of amino acids in the given beta-sheet or alpha-helix to eliminate the differences caused by non-equal length of the sheet or helix. Hydrophobicity units obtained have been averaged according to the number of internal (all beta-strands and helix alpha 7) and external (helices alpha 1-alpha 6 and alpha 8) elements of secondary structure of the alpha-amylase (beta alpha)8 barrel. The averaged hydrophobicity units have been found to correlate with the thermal stability of the three Bacillus alpha-amylases in terms of the increased hydrophobicity of the interior as well as the increased hydrophilicity of the exterior of the (beta alpha)8 barrel domain for the alpha-amylase with increased thermostability.

Bacillus↗
PubMed1990

Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain.

A recombinant plasmid (pMK57) was constructed by cloning the Bacillus stearothermophilus alpha-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed into Escherichia coli strain JM 103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced alpha-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more alpha-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much alpha-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.

Cloning, Molecular↗
PubMed1991

Conjugal transfer of recombinant transposon Tn916 from Escherichia coli to Bacillus stearothermophilus.

A gene for thermostable amylase has been inserted at the BstXI site of Tn916. Mating experiments demonstrated that unlike Tn916, the recombinant transposon, designated Tn916A, could transfer from Escherichia coli to Bacillus stearothermophilus BR219 in broth matings, resulting in chromosomal integration of the transposon and expression of the amylase at significant levels.

Cloning, Molecular↗
PubMed1991

Plasmids in Bacillus stearothermophilus coding for bacteriocinogeny and temperature resistance.

Obligately thermophilic strains of Bacillus stearothermophilus were screened for the presence of plasmids by agarose gel electrophoresis. All strains in our collection contained large plasmids (20 x 10(6)-80 x 10(6)) and were divided into four groups with respect to their plasmid pattern and production of bacteriocins. The major plasmid species were designated pSE407 (38.7 x 10(6)), pSE409 (29.0 x 10(6)), pSE411 (21.5 x 10(6)), and pSE410 (23.5 x 10(6)). Their physical endonuclease maps were constructed, and by Southern blots and hybridizations it was shown that these plasmids were related. From curing experiments and electrotransformations (electroporations) we conclude that pSE407, pSE410, and pSE411 code for temperature resistance. In addition pSE410 codes for bacteriocin production and resistance. Plasmid pSE409 probably also codes for bacteriocin production and resistance.

Bacteriocins↗