Nucleotide sequence of the triose phosphate isomerase gene of Escherichia coli

Summary We report here the complete nucleotide sequence of the E. coli triose phosphate isomerase gene. The gene encodes a polypeptide of 255 amino acids which is approximately 46% homologous to eukaryotic triose phosphate isomerases, and approximately 38% homologous to the enzyme from a thermophilic bacterium, Bacillus stearothermophilus. The nucleotide sequence is 55% homologous to that of the corresponding gene in the yeast Saccharomyces cerevisiae. To our knowledge, this is the first report of the sequence of a gene coding a glycolytic enzyme from a prokaryotic organism.     

Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices

Amino acid sequences have been compared for thermophilic and mesophilic molecules from six different protein families, which include lactate and glyceraldehyde-3-phosphate dehydrogenases, triose phosphate isomerases, superoxide dismutases, thermolysins and subtilisins. Since a three-dimensional structure was known for at least one of the sequences in each family, analysis of preferred residue substitutions, presumably to achieve thermal stability, could be examined from a structural context. The overall results, which are generally consistent across all the families, suggested decreased flexibility and increased hydrophobicity in alpha-helical regions as the main stabilizing principles. The most favoured residual exchanges, hopefully useful in engineering stability into proteins, are discussed.     

Evidence for filaggrin as a component of the cell envelope of the newborn rat.

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.     

Sites of methyl esterification and deamination on the aspartate receptor involved in chemotaxis

The receptors involved in bacterial chemotaxis are post-translationally modified by specific enzymes which catalyze the deamination of glutaminyl residues and the methyl esterification and demethylation of glutamyl residues. In this work we identify the sites of these covalent modifications on the aspartate receptor from Salmonella typhimurium. These were identified using the properties of the Staphylococcus aureus V8 protease which cleaves peptide bonds following glutamyl but not glutaminyl residues. We show here that bonds following methyl-esterified glutamyl residues are also resistant to the protease. A comparison of the fragments obtained after V8 protease cleavage of methyl-esterified (or deaminated) peptides with the fragments from the corresponding unmodified peptides immediately yields the sites of modification. Three of the four methyl-esterified glutamyl residues are located near the middle of the receptor amino acid sequence; one of these is synthesized as a glutaminyl residue and is deaminated by the esterase to form a glutamyl residue. The fourth site of methyl esterification is located near the carboxyl terminus. All four sites occupy analogous positions in a well-conserved arrangement of residues which may form a binding site for the esterase and the methyltransferase.     

The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate

1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.     

Disequilibrium in the triose phosphate isomerase system in rat liver

1. The equilibrium constant at 38 degrees and I 0.25 of the triose phosphate isomerase reaction was found to be 22.0 and that of the aldolase reaction, 0.99x10(-4)m. The [dihydroxyacetone phosphate]/[glyceraldehyde phosphate] ratio was found to be 9.3 in rat liver. The causes of the apparent deviation of the triose phosphate isomerase system from equilibrium in vivo have been investigated. 2. The equilibria of the triose phosphate isomerase and aldolase reactions were studied with relatively large concentrations of crystalline enzymes and small concentrations of substrates, approximating to those found in rat liver and muscle. There was significant binding of fructose diphosphate by aldolase under these conditions. There was no evidence that binding of glyceraldehyde phosphate by either enzyme affected the equilibria. 3. The deviation from equilibrium of the triose phosphate isomerase system in rat liver can be accounted for by the low activity of the enzyme, in relation to the flux, at low physiological concentrations of glyceraldehyde phosphate (about 3mum). It has been calculated that a flux of 1.8mumoles/min./g. wet weight of liver would be expected to cause the measured degree of disequilibrium found in vivo. 4. The conclusion that the triose phosphate isomerase is not at equilibrium is in accordance with the situation postulated by Rose, Kellermeyer, Stjernholm & Wood (1962) on the basis of isotope-distribution data. 5. The triose phosphate isomerase system is closer to equilibrium in resting muscle probably because of a very low flux and a high enzyme concentration. 6. The aldolase system deviated from equilibrium in rat liver by a factor of about 10 and by a much greater factor in resting muscle. 7. The measurement of total dihydroxyacetone phosphate and glyceraldehyde phosphate content indicates the concentrations of the free metabolites in the tissue. This may not hold for fructose diphosphate, a significant proportion of which may be bound to aldolase.     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.     

Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate

The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C₃ species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.Abbreviations DHAP Dihydroxyacetonephosphate - Fru1,6-bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - PEP(Case) phosphoenolpyruvate (carboxylase) - PGA 3-phosphoglycerate - Rubisco ribulose-1,5-bisphosphate carboxylase     

The magnesium protein interaction in nucleotide complexes of phos-phoglycerate kinase

The X-ray structure determination of yeast phosphoglycerate kinase and subsequent substrate binding studies have helped to define the binding sites for the triose and nucleoside phosphate substrates. This communication deals with one feature of the binding site—the location of an aspartic acid residue close to the phosphoryl binding site of the nucleotide substrate—and relates this and other structural features of the active site to the properties of this enzyme as deduced from nuclear magnetic resonance studies.     

Evidence for an essential glutamyl residue in yeast hexokinase.

Yeast hexokinase is rapidly inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and nitrotyrosyl ethyl ester. Sugar substrates afford a partial protection, which is increased by the addition of ADP. Inactivation of the enzyme takes place concomitantly with the incorporation of 1 mol of nitrotyrosine per mol of 50 000-dalton subunit. Exhaustive proteolytic digestion of the modified protein and isolation of the nitrotyrosyl peptide by affinity chromatography, followed by electrophoresis, lead to the identification of the modified residue as a glutamyl residue. This modification of hexokinase occurs without gross conformational changes. The enzyme still binds its substrates, though binding of the nucleotides is perturbed. While the substrates afford a partial protection, they increase the incorporation of nitrotyrosine ethyl ester into the enzyme. This may be attributed to local conformational changes which their binding induces. It is concluded that a glutamyl residue is essential for yeast hexokinase activity and its catalytic function is discussed.     

Effect of the modification of the processing site of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase on the production of active enzyme by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cells

A site-directed modification at position P1 of the processing site of Bacillus intermedius glutamyl endopeptidase was carried out. Variants of the protease gene were obtained that correspond to the protein with an Ala, Asn, Ser, or Glu residue at this position substituted for the Lys residue. The residue in the P1 position of the processing site was shown to affect substantially the production of the active enzyme; however, none of the mutations leads to the complete termination of active protein production by cells.     

A rapid method for measuring organelle-specific substrate transport in homogenates from plant tissues

A rapid method is described for measuring organelle-specific metabolite transport systems in crude homogenates from plants. The tissues were homogenized in liquid nitrogen, extracted with buffer and reconstituted into artificial membranes. The method allowed demonstration of the known different substrate specificities of chloroplast triose phosphate/phosphate translocators from C₃- and C₄-plants, of the triose phosphate/phosphate translocator from non-green tissue, and of the dicarboxylate translocator. It thus by-passes the necessity to isolate intact plant organelles and, in addition, only a low amount of tissue material is required for transport measurements.Abbreviations Chl chlorophyll - TPT triose phosphate/phosphate translocator Dedicated to Professor F.-C. Czygan on the occasion of his 60th birthdayThis research was supported by the Bundesministerium für Forschung und Technologie. A.W. is a recipient of a Ph.D. fellowship from the Deutsche Forschungsgemeinschaft.     

Evidence for the activity of immobilised monomers of triose phosphate isomerase.

Chicken muscle triose phosphate isomerase was immobilised by attachment to Sepharose 4B. The immobilised dimeric enzyme was dissociated with guanidinium chloride to yield bound monomeric triose phosphate isomerase. This regained activity on removal of the denaturant, showing that isolated monomers possess activity; the apparent K_m of the immobilished subunits was the same as that of the immobilised dimers. Under appropriate conditions, it was possible to rehybridise the immobilised monomers to native dimers, and also to form a hybrid dimer from the chicken muscle and rabbit muscle enzymes.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Elementary modes analysis of photosynthate metabolism in the chloroplast stroma.

We briefly review the metabolism of the chloroplast stroma, and describe the structural modelling technique of elementary modes analysis. The technique is applied to a model of chloroplast metabolism to investigate viable pathways in the light, in the dark, and in the dark with the addition of sedoheptulose-1,7-bisphosphatase (normally inactive in the dark). The results of the analysis show that it is possible for starch degradation to enhance photosynthetic triose phosphate export in the light, but the reactions of the Calvin cycle alone are not capable of providing a sustainable flux from starch to triose phosphate in the dark. When reactions of the oxidative pentose phosphate pathway are taken into consideration, triose phosphate export in the dark becomes possible by the operation of a cyclic pathway not previously described. The effect of introducing sedoheptulose-1,7-bisphosphatase to the system are relatively minor and, we predict, innocuous in vivo. We conclude that, in contrast with the traditional view of the Calvin cycle and oxidative pentose phosphate pathway as separate systems, they are in fact, in the context of the chloroplast, complementary and overlapping components of the same system.     

Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.

We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.     

Competitive inhibitors of Mycobacterium tuberculosis ribose-5-phosphate isomerase B reveal new information about the reaction mechanism

Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (K(m) 57 microm) and 4-phospho-d-erythronate (K(i) 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.     

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.