PCR site-directed mutagenesis using Pyrococcus sp GB-D polymerase coupled to a rapid screening procedure. Application to a beta-glucanase gene.
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Archaea↗
Biomedical subjects
A Planas
Publications and source records attributed to A Planas.
A specific chromophoric substrate for activity assays of 1,3-1,4-beta-D-glucan 4-glucanohydrolases.
The synthesis of 4-methylumbelliferyl 3-beta-O-cellobiosyl-beta-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-beta-D-glucan 4-glucanohydrolases are described. The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose beta-D-glucan polysaccharide is degraded down to a tri- and tetrasaccharide by an extracellular extract of recombinant E. coli expressing and secreting Bacillus licheniformis 1,3-1,4-beta-glucanase. The trisaccharide 1 is further chemically transformed into the title compound. Its use as substrate for an enzyme activity assay, the specificity of cleavage, and kinetic parameters are reported. As it undergoes a single glycosidic bond hydrolysis with release of 4-methylumbelliferone, direct UV monitoring of the reaction provides a sensitive kinetic assay of the enzyme action.
Biotechnology↗
Design and chemoenzymatic synthesis of thiooligosaccharide inhibitors of 1,3:1,4-beta-D-glucanases.
A successful chemoenzymatic synthesis of oligosaccharides with an interglucosidic sulfur atom as inhibitors of 1,3:1,4-D-glucanases is described. The key compound 3a was synthesized from acetylated 1-thio-beta-laminaribiose 4 and the methyl 4'-O-triflyl-lactoside 5. After de-O-acylation, the tetrasaccharide 3b was used as an acceptor and glucose-1-P as a donor in a phosphorolytic elongation catalysed by cellodextrin phosphorylase from Clostridium thermocellum. The expected pentasaccharide 2a and hexasaccharide 1 were isolated in 56% and 13% yield, respectively. As expected, the thiooligosaccharides 1, 2a, and 3b were resistant to enzymatic cleavage by 1,3:1,4-beta-D-glucanase isolated from Bacillus licheniformis. Furthermore, they have been shown to act as competitive inhibitors of the hydrolysis of the chromophoric trisaccharide substrate 11 by this enzyme.
Binding Sites↗
[Utilization of inhaled nitric oxide in a case of respiratory insufficiency following lung transplantation].
Pulmonary hypertension and transient graft dysfunction may complicate the immediate postoperative course of patients undergoing lung transplantation. We report the effect of inhaled nitric oxide (NO) in a patient with pulmonary edema and severe hypoxemia in the period following bilateral lung transplantation. NO was delivered through the inspiratory line and its mean concentration was monitored in the trachea. PaO2/FiO2 improved from 65 to 105 with an NO concentration of 1 ppm, but no further improvement was obtained by increasing the dose to 5 ppm. Pulmonary vascular resistance (PVR) decreased from a baseline value of 251 dynes-sec/cm5 to a low of 213 dynes-sec/cm5 with NO administration. A slight increase in PVR seems to have been the main factor limiting the therapeutic efficacy of inhaled NO in this case.
Administration, Inhalation↗
Crystal structure of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase at 1.8 A resolution.
The crystal structure of the 1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis is solved at a resolution of 1.8 A and refined to R = 16.5%. The protein has a similar beta-sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16-M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3-1,4-beta-D-glucan 4-glucanohydrolases.
Bacillus↗
Synthesis of 4-methylumbelliferyl-beta-D-glucan oligosaccharides as specific chromophoric substrates of (1-->3), (1-->4)-beta-D-glucan 4-glucanohydrolases.
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Carbohydrate Sequence↗
Transforming growth factor-alpha immunoreactivity in the developing and adult brain.
Transforming growth factor-alpha immunoreactivity is examined in the developing and adult brain of cats and rats, and in the adult human brain in cryostat sections immediately processed free-floating with a well-characterized monoclonal antibody which does not cross-react with epidermal growth factor. Transforming growth factor-alpha immunoreactivity is observed in neurons of the cerebral neocortex, subiculum, hippocampus, striatum, thalamus, amygdala, basal forebrain, mesencephalon, cerebellar cortex, dentate nucleus and brainstem during development and in adulthood. The intensity of the immunoreaction directly correlates with the size of the cytoplasm. Diffuse transforming growth factor-alpha immunoreactivity also occurs in the white matter of the cerebrum, cerebellum and brainstem in the kitten, but not in the adult cat. In addition to neurons, numbers of glial cells in the cerebellar white matter, brainstem and cerebral hemispheres during development, and a few glial cells in the cerebellar cortex, diencephalon, cerebral cortex and white matter in adults are strongly transforming growth factor-alpha immunoreactive. These results support the concept that transforming growth factor-alpha is widely distributed in the brain of mammals, localizes in both neurons and glial cells, and is development dependent. These findings also suggest that transforming growth factor-alpha may play a role in the developing and adult central nervous system.
Adult↗
Contribution of a disulfide bridge to the stability of 1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis.
Bacillus 1,3-1,4-beta-glucanases possess a highly conserved disulfide bridge connecting a beta-strand with a solvent-exposed loop lying on top of the extended binding site cleft. The contribution of the disulfide bond and of both individual cysteines (Cys61 and Cys90) in the Bacillus licheniformis enzyme to stability and activity has been evaluated by protein engineering methods. Reduction of the disulfide bond has no effect on kinetic parameters, has only a minor effect on the activity-temperature profile at high temperatures, and destabilizes the protein by less than 0.7 kcal/mol as measured by equilibrium urea denaturation at 37 degrees C. Replacing either of the Cys residues with Ala destabilizes the protein and lowers the specific activity. C90A retains 70% of wild-type (wt) activity (in terms of Vmax), whereas C61A and the double mutant C61A-C90A have 10% of wt Vmax. A larger change in free energy of unfolding is seen by equilibrium urea denaturation for the C61A mutation (loop residue, 3.2 kcal/mol relative to reduced wt) as compared with the C90A mutation (beta-strand residue, 1.8 kcal/mol relative to reduced wt), while the double mutant C61A-C90A is approximately 0.8 kcal/mol less stable than the single C61A mutant. The effects on stability are interpreted as a result of the change in hydrophobic packing that occurs upon removal of the sulfur atoms in the Cys to Ala mutations.
Bacillus↗
[Anesthesia in a patient with latex allergy].
Anaphylactic reactions triggered by latex-based products are increasingly frequent, particularly in the hospital environment. We describe a patient with a prior history of atopic allergy and documented allergy to latex who was scheduled for abdominal surgery. A great deal of anesthetic and surgical equipment contains latex and substitutes for such material must be used in order to prevent severe hypersensitivity reactions.
Adrenal Cortex Hormones↗
[Special anesthesia care in heart transplantation. The management of right ventricular failure].
Right ventricular dysfunction is a common clinical event after heart transplantation. The major cause is the failure of right ventricle (RV) to adapt to the pulmonary hypertension (PH) secondary to chronic heart failure. Nevertheless, this dysfunction is usually transient owing to the reversibility of PH, the nature of which is mainly passive. Therefore, it is particularly important to perform a preoperative hemodynamic study to identify those cases in which PH is a permanent component, a situation that excessively increases the risk of postoperative RV failure. Once this occurs, the treatment is complex. The available therapeutic measures include the use of vasodilators such as prostaglandin E1 and nitric oxide.
Anesthesia↗
Naturally occurring cell death in the developing cerebral cortex of the rat. Evidence of apoptosis-associated internucleosomal DNA fragmentation.
Naturally occurring dead cells in the developing rat neocortex, subcortical white matter and hippocampus, which increase in number during the first postnatal week and decrease thereafter to disappear by the end of the first month, were examined by in situ labeling of nuclear DNA fragmentation. These cells showed peripheral chromatin condensation or extremely dark, often fragmented, nuclei. Southern hybridization following agarose gel electrophoresis of DNA extracted from the developing cortex, but not from adult brain, showed a 'ladder' pattern which is typical of internucleosomal DNA fragmentation. Taken together these results show that naturally occurring cell death (programmed cell death) in the developing cerebral cortex has the morphology of apoptosis and is associated with endonuclease activation.
Aging↗
Increased expression of bcl-2 immunoreactivity in the developing cerebral cortex of the rat.
Bcl-2 proto-oncogene encodes a protein which may cancel the cell death programme in normal development and experimentally induced conditions. Strong bcl-2 immunoreactivity occurs in the neocortex and hippocampus of the developing rat during the 1st postnatal week. Bcl-2 immunoreactivity rapidly decreases from this age onwards to steady very low levels in adulthood. Since increased expression of bcl-2 immunoreactivity during cortical neurogenesis is coincidental in time with a special vulnerability of cortical neurons to naturally occurring cell death, it is suggested that bcl-2 may have a role in regulating cell death and survival during cortical morphogenesis.
Animals↗
Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis.
Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis. Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK alpha of the catalytic residues.
Amino Acid Sequence↗
Improved efficiency in site-directed mutagenesis by PCR using a Pyrococcus sp. GB-D polymerase.
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DNA-Directed DNA Polymerase↗
Stereochemical course and structure of the products of the enzymic action of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis.
The stereochemical course of the reaction catalysed by endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) has been determined by 1H n.m.r. The enzyme-catalysed hydrolysis of barley beta-glucan proceeds with overall retention of the anomeric configuration, indicating that the enzyme operates through a double-displacement mechanism. The structures of the final oligosaccharide products, 3-beta-O-cellobiosyl D-glucopyranoside and 3-beta-O-cellotriosyl D-glucopyranoside, have been completely assigned by 1H- and 13C-n.m.r. spectroscopy.
Bacillus↗
Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis.
Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinant E. coli and their kinetics analysed with barley beta-glucan. Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.
Amino Acid Sequence↗
Prediction and Fourier transform infrared spectroscopy estimation of the secondary structure of a Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase.
The secondary structure of a recombinant Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase (EC.3.2.1.73) has been estimated by Fourier Transform Infrared Spectroscopy and also predicted by the algorithm of Chou and Fasman. From the curve fitting of the deconvolved IR spectrum, the most probable distribution of the secondary structural classes appears to be about 40% beta-sheet, 25% reverse turn, 24% non-ordered and 11% alpha-helix. From theoretical prediction of secondary structure the protein would present 37% beta-sheet, 31% reverse turn, 22% non-ordered and 10% alpha-helix.
Bacillus↗
Contribution to catalysis and stability of the five cysteines in Escherichia coli aspartate aminotransferase. Preparation and properties of a cysteine-free enzyme.
The five cysteines, at positions 82, 191, 192, 270, and 401, of Escherichia coli aspartate aminotransferase (AATase) were, individually and in some combinations, converted to alanine by site-directed mutagenesis (C82A, C191A, C192A, C270A, C401A). Cys-191, which is conserved in all AATase isozymes, was mutated to serine as well (C191S). A quintuple mutant, with all cysteines converted to alanines (Quint), was also constructed. The effects of these single and multiple mutations were examined by steady-state kinetics and urea denaturation. The thermal stabilities of Quint and of the wild-type enzyme (WT) were determined by differential scanning calorimetry. The mutants had kcat values up to 50% greater than that of WT and KMAsp and KM alpha-KG values up to 1.5- and 3.3-fold higher than that of WT. The mutants C82A and C191A exhibit nearly the same CM in urea denaturation experiments as WT, while the other single mutants and Quint are less stable, with CM differences of up to 0.7 M urea. Quint is also less thermostable than WT, with a delta TM of 3.3-4.4 degrees C. Thus the five cysteine replacements yield small, but significant, changes in catalytic and denaturation parameters, but none of the cysteines was found to be essential. The changes manifested in the mutation of the conserved Cys-191 to alanine are no greater than those observed with the four nonconserved cysteines. We consider the evolutionary implications of these findings.
Amino Acid Sequence↗