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At least 991 records · Page 55Linked to original sources
PubMed1992

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.

Alanine↗
PubMed2004

The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes.

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen.

Animals↗
PubMed2001

Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates.

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 +/- 14 microM for L1 and 24.8 +/- 3.8 microM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activity preparations of E1, the Km values were > or =160 microM for L1 and > or =35 microM for pyruvate and k(cat) was > or =70 s(-1). Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (Km > or =15 mM, k(cat) approximately equals 5 s(-1); k(cat)/Km decreased >1,500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha-keto acid dehydrogenases.

Acetylation↗
PubMed2000

Influence of transketolase substrates on its conformation.

Dynamics stimulation of the holotransketolase molecule revealed that the enzyme's conformation in crystal was different from that in solution. It was shown also that dissolved holotransketolase can bind aldose (the acceptor substrate) even in the absence of ketose (the donor substrate). The holotransketolase conformation did not change upon aldose binding unlike in the case of ketose binding/cleavage. Therefore the conformation of a catalytic complex of holotransketolase with an intermediate-i.e., a glycolaldehyde residue formed upon binding and subsequent cleavage of ketose-differed, at least in solution, from the conformation of both the free and aldose-complexed holotransketolase. Some structural peculiarities of the holotransketolase with the intermediate were established by means of molecular dynamics stimulation.

Acetaldehyde↗
PubMed2002

Spectroscopic evidence for participation of the 1',4'-imino tautomer of thiamin diphosphate in catalysis by yeast pyruvate decarboxylase.

The 1',4'-iminopyrimidine tautomeric form of the coenzyme thiamin diphosphate (ThDP), implicated in catalysis on the basis of the conformation of enzyme-bound ThDP, has been observed by both ultraviolet absorption and circular dichroism spectroscopy. On yeast pyruvate decarboxylase, the unusual tautomer is observed in an active center variant in which catalysis in the post-decarboxylation regime of the reaction is compromised. In a model system consisting of N1-methyl-4-aminopyrimidinium or N1-methyl-N4-n-butylpyrimidinium salts, on treatment with either NaOH in water, or DBU in DMSO there is an intermediate formed with lambda(max) near 310 nm, and this intermediate reverts back to the starting salt on acidification. Proton NMR chemical shifts are consistent with the intermediate representing the 1-methyl-4-imino tautomer. On the enzyme, the intermediate could be observed by rapid-scan stopped flow with UV detection when reacting holoenzyme of the E477Q active center variant with pyruvate, and by circular dichroism even in the absence of pyruvate. This represents the first direct observation of the imino tautomeric form of ThDP both on the enzyme and in models, although some years ago, this laboratory had already reported some pertinent acid-base properties for its formation [Jordan, F., and Mariam, Y. H. (1978) J. Am. Chem. Soc.100, 2534-2541]. The work also represents the first instance in which a rare tautomer implicated in catalysis is identified and suggests that such tautomeric catalysis may be more common in biology than hitherto recognized.

Acetaldehyde↗
PubMed1996

A human homolog of bacterial acetolactate synthase genes maps within the CADASIL critical region.

CADASIL, a recently identified autosomal dominant condition characterized by the recurrence of subcortical infarcts leading to dementia, was previously mapped to chromosome 19p13.1 within a 2-cM interval, D19S226-D19S199. No recombination event was observed with D19S841, a highly polymorphic microsatellite marker isolated from a cosmid mapped to this region. We recently identified within this cosmid a conserved sequence that we used to screen a fetal brain cDNA library and isolated an ubiquitous and abundantly transcribed gene. We did not detect any mutation of this gene in CADASIL patients, suggesting that it is not implicated in this disorder. Interestingly, this gene encodes a putative protein homologous to several thiamine pyrophosphate-binding proteins previously identified in bacteria, yeast, and plants. The proteins with the highest degree of similarity were the acetolactate synthase enzymes which, in prokaryotes, are involved in the branched chain amino acid biosynthetic pathway, raising fascinating questions on the yet unknown function of this gene in mammals.

Acetolactate Synthase↗
PubMed1994

The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum.

The crystal structure of pyruvate oxidase (EC 1.2.3.3) from Lactobacillus plantarum stabilized by three point mutations has been refined at 2.1 A resolution using the simulated annealing method. Based on 87,775 independent reflections in the resolution range 10 to 2.1 A, a final R-factor of 16.2% was obtained at good model geometry. The wild-type enzyme crystallizes isomorphously with the stabilized enzyme and has been analyzed at 2.5 A resolution. Pyruvate oxidase is a homotetramer with point group symmetry D2. One 2-fold axis is crystallographic, the others are local. The crystallographic asymmetric unit contains two subunits, and the model consists of the two polypeptide chains (residues 9 through 593), two FAD, two ThDP*Mg2+ and 739 water molecules. Each subunit has three domains; the CORE domain, the FAD domain and the ThDP domain. The FAD-binding chain fold is different from those of other known flavoproteins, whereas the ThDP-binding chain fold resembles the corresponding folds of the two other ThDP enzymes whose structure is known, transketolase and pyruvate decarboxylase. The peptide environment most likely forces the pyrimidine ring of ThDP into an unusual tautomeric form, which is required for catalysis. The structural differences between the wild-type and the stabilized enzyme are small. All three point mutations are at or near to the subunit interfaces, indicating that they stabilize the quarternary structure as had been deduced from reconstitution experiments.

Amino Acid Sequence↗
PubMed1994

Refined structure of transketolase from Saccharomyces cerevisiae at 2.0 A resolution.

The crystal structure of transketolase from Saccharomyces cerevisiae has been refined to a crystallographic residual of 15.7% at 2.0 A resolution using the program package X-PLOR. The refined model of the transketolase homodimer, corresponding to 1356 amino acid residues in the asymmetric unit, consists of 10,396 protein atoms, 1040 solvent molecules, 52 thiamine diphosphate atoms and two calcium ions. All amino acid residues except for the two N-terminal residues of the two subunits are defined in the electron density maps and refined. The estimated root-mean-square (r.m.s.) error of the model is less than 0.2 A as deduced from Luzzati plots. The r.m.s. deviation from ideality is 0.017 A for bond distances and 3.1 degrees for bond angles. The main-chain torsion angles of non-glycine residues lie within the allowed regions of the Ramachandran plots. The model shows a very good fit to the electron density maps. The average B-factor for all protein atoms in the first subunit is 19 A2, and 15A2 in the second. The average B-factor for solvent atoms is 32A2. The two subunits of transketolase were refined independently and have nearly identical structures with an r.m.s. deviation of 0.24 A for C alpha atoms 3 to 680, and slightly less when aligning the individual domains. A few exceptions from the 2-fold symmetry are found, mostly in the surface residues. The thiamine diphosphate cofactors have identical conformations. The cofactor is shielded from solvent except for the C-2 atom of the thiazolium ring. A calcium ion is bound to the diphosphate group of thiamine and protein ligands. The metal binding site and the interactions of thiamine diphosphate with protein residues are described. A network of hydrogen bonds consisting of glutamic acid residues and internal water molecules connects the two thiamine diphosphate molecules. Its structure and possible functional implications are discussed.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution.

The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4' position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring.

Amino Acid Sequence↗
PubMed2002

Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors.

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides.

Acetolactate Synthase↗
PubMed1993

The influence of the effectors of yeast pyruvate decarboxylase (PDC) on the conformation of the dimers and tetramers and their pH-dependent equilibrium.

The influence of effectors of yeast pyruvate decarboxylase, phosphate, pyruvamide, thiamin diphosphate and Mg++, on the pH-dependent equilibrium between dimers and tetramers was studied by synchrotron radiation X-ray solution scattering. Thiamin diphosphate and phosphate shift the equilibrium to higher pH values without altering the structure of the oligomers. Pyruvamide, a substrate analogue activator, induces a significant change in the structure of the tetramer. By eliminating radiation damage by addition of dithioerythrol to the buffers, the scattering curves could be measured accurately over a large angular range. They were expanded in terms of spherical harmonics to obtain the shapes of the dimers and tetramers with higher resolution than was hitherto possible. This also allowed us to position the dimers, which are centrosymmetric at low resolution, in the tetramers which have 222 symmetry. The results indicate that addition of pyruvamide results in a less compact tetramer owing to structural changes in the dimers and to their displacements.

Biophysical Phenomena↗
PubMed1975

The fine structure and phosphatase cytochemistry of the golgi complex and associated structures in the sertoli cells of Syrian hamsters.

The Golgi complex in the Sertoli cell of the Syrian hamster is well developed and consists of stacks of cisternae and associated vesicles. The inner- and outermost cisternae of the Golgi stacks are usually moderately dilated and exhibit numerous fenestrations. The middle portions of the intermediate cisternae are greatly flattened and not fenestrated, but toward the periphery these cisternae gradually become dilated and show a few fenestrations. On the inner aspect of the Golgi stacks the following structures are seen frequently: (1) one or two series of linearly arrayed circular profiles some of which are interconnected by tubules; (2) networks of anastomosing tubules with circular or oval meshes (800 to 1200 A in diameter); and/or (3) irregularly disposed tubules. The circular profiles and tubules are approximately 450 A in diameter. Acid phosphatase activity was localized in these anastomosing tubules when the tissues were incubated for more than one hour in a modified Gomori's medium (Barka and Anderson, 1963). Strong thiamine pyrophosphatase activity was demonstrated in the inner one to three cisternae of the Golgi stacks but not in the associated tubules. The system of the Golgi associated tubules is morphologically and histochemically distinct from the Golgi stacks and is probably equivalent to the Golgi-endoplasmic reticulum-lysosome system (GERL) in other cell types. The three dimensional aspects of the GERL-equivalent system are discussed.

Acid Phosphatase↗
PubMed1975

Aspects of furrow membrane formation in the cleaving Drosophila embryo.

During blastoderm formation, the Drosophila embryo produces a large area of new membrane to accommodate the simultaneous demands of approximately four thousand newly cleaved cells. The embryo was examined with the electron microscope at various stages during cleavage in order to investigate the high membrane forming capacity of these cells. Embryos were subjected to the histochemical procedure for the demonstration of thiamine pyrophosphatase. The enzyme was present in the cisternae of the endoplasmic reticulum, Golgi bodies and the nuclear envelope. No activity could be demonstrated on the furrow surface or at the furrow tip despite closely adjacent reactive cisternae. It is concluded that the endoplasmic reticulum and Golgi bodies are not major contributors to the new surface. Lamellar bodies were frequently observed in the cytoplasm of all stages examined. The bodies showed a lamellar periodicity of approximately 3.5 nm, and were ultrastructurally similar in appearance after a variety of fixation procedures. The distribution of these bodies was markedly related to the stage of blastoderm formation. Before the commencement of cleavage, lamellar bodies were very prominent within a region 4--5 mum below the cell surface. As cleavage progressed, the bodies became sparse or absent from this region but were apparent at the base of the blastoderm cells or in the sub-blastoderm region, where they were not previously present. Lamellar bodies with leaflets closely associated with, or in apparent continuity with, the cleavage furrow membrane were frequently observed. In these regions the lamellar periodicity was the same as the thickness of the membrane laminae. It is suggested that these bodies play a role in the synthesis of new membrane in the furrow. Intercellular contact specializations between the developing membranes of the furrow were restricted to incipient desmosones and point contacts where the intercellular gap was reduced to 3 nm or less.

Animals↗
PubMed1988

Thiamine status after major trauma.

A prospective study of thiamine status in patients with severe injuries (injury severity scores greater than 12) showed that they all developed signs of severe thiamine deficiency on the basis of transketolase activity, the standard biochemical test for thiamine deficiency. This occurred within the first week after their injuries despite routine enteral or parenteral feeding including vitamin supplements. This may be due to increased requirements secondary to increased metabolic activity or increased cellular losses. An increased awareness of thiamine requirements and metabolism is essential to avoid the appearance of deficiency syndromes.

Adult↗