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PubMed2005

High-pressure acceleration of the growth kinetics of glucose isomerase crystals.

The growth and dissolution rates of glucose isomerase crystals ({1 0 1} face) were measured in situ at 0.1 and 100 MPa. From these data, we determined that the solubilities at 25 degrees C were C(e) = 3.1 +/- 0.9 and 2.6 +/- 0.5 mg mL(-1) at 0.1 and 100 MPa, respectively. At the same supersaturation of sigma = 2.5 (sigma identical with ln(C/C(e)), C = the concentration of glucose isomerase, C(e) = the solubility) and temperature (T = 25 degrees C), the growth rate under 100 MPa was 7.6 times larger than that under 0.1 MPa. This result shows, for the first time, a kinetic acceleration of the growth rates of protein crystals with increasing pressure. The growth rates vs sigma data fitted well with a two-dimensional nucleation growth model of a polynucleation type. The fitting results indicate that the acceleration is mainly due to the decrease in the molecular surface energy of the glucose isomerase crystal with pressure.

Aldose-Ketose Isomerases↗
PubMed1998

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana.

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.

Amino Acid Sequence↗
PubMed1998

Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: purification and molecular analysis of the enzyme.

Purification and molecular analysis of ribose-5-phosphate isomerase (EC 5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzyme was enriched from a haploid deletion mutant containing the wild-type gene on a multicopy plasmid elaborating the following steps: ammonium sulphate precipitation, interfacial salting out on Sepharose 6B, high performance liquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. The enzyme activity was found to be rather unstable possibly caused by removal of stabilizing cofactors or proteins during the purification procedure. The purified enzyme showed a hyperbolic dependence on the substrate ribose-5-phosphate with a K(m)-value of 1.6 +/- 0.3 mmol/l. For the native enzyme a molecular mass of 115 +/- 10 kDa was determined as found by saccharose density gradient centrifugation, sedimentation equilibrium analysis, size exclusion chromatography and polyacrylamide gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting revealed one band with a molecular mass of 31 +/- 2 kDa. Thus, the native enzyme is composed of four subunits of identical size. The molecular mass of the subunit and the identified N-terminal sequence of 33 amino acids fits well the 258 amino acid protein encoded by the S. cerevisiae RKI open reading frame, which was characterized previously only by increasing specific activities of ribose-5-phosphate isomerase in cells after cloning the gene. On the basis of the conserved amino acids an alignment of the amino acid sequence of ribose-5-phosphate isomerase from yeast with those of the enzyme from mouse, spinach and Escherichia coli is presented.

Aldose-Ketose Isomerases↗
PubMed2001

Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate<-->dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved.

Adenosine Triphosphate↗
PubMed1973

The deoxyfluoro-D-glucopyranose 6-phosphates and their effect on yeast glucose phosphate isomerase.

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.

Computers↗
PubMed2004

Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins.

Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al. (1994) EMBO J. 13, 3245-3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI. By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.

Amino Acid Motifs↗
PubMed1983

Induction of glycolytic enzyme synthesis in proliferating fibroblasts. Study of phosphofructokinase, glucose phosphate isomerase and pyruvate kinase.

Specific activity of phosphofructokinase is 7-8-fold higher in exponentially growing human fibroblasts than in quiescent cells, but the difference is considerably less pronounced for two other glycolytic enzymes, glucose phosphate isomerase and pyruvate kinase. The ratio of the F-type to L-type phosphofructokinase subunits is essentially the same in growing and resting cells, 4:1. F-type-phosphofructokinase-related antigen concentration is decreased in resting cells as compared with proliferating fibroblasts, but relatively less than the enzyme activity; the ratio of the enzyme activity to the antigen concentration (immunological specific activity) is therefore lower in resting than in growing fibroblasts. Synthesis of phosphofructokinase, as a percentage of the total protein synthesis, is about 30-fold greater during the proliferative phase than in quiescent cells, but this difference is only 3-4-fold for glucose phosphate isomerase and pyruvate kinase. Modulation of the synthesis of phosphofructokinase therefore seems to be responsible for the changes of its specific activity in function of cell proliferation. The appearance of some inactive cross-reacting material in quiescent cells is probably due to post-translational alteration of the pre-synthesized molecules. Compared with other glycolytic enzymes, such as glucose phosphate isomerase and pyruvate kinase, phosphofructokinase seems to be the (or one of the) preferential target of glycolytic induction in proliferating cells.

Cell Division↗
PubMed1991

D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Gene cloning, sequence and expression.

Arthrobacter strain N.R.R.L. B3728 superproduces a D-xylose isomerase that is also a useful industrial D-glucose isomerase. The gene (xylA) that encodes it has been cloned by complementing a xylA mutant of the ancestral strain, with the use of a shuttle vector. The 5' region shows strong sequence similarity to Escherichia coli consensus promoters and ribosome-binding sequences and allows high levels of expression in E. coli. The coding sequence shows similarity to those for other D-xylose isomerases and is followed by 22 nucleotide residues with stop codons in each reading frame, a good 'consensus' ribosome-binding site and an open reading frame showing similarity to those of known D-xylulokinases (xylB). Studies on the expression of the cloned gene in Arthrobacter and in E. coli suggest that the two genes are part of a xyl operon regulated by a repressor that is defective in strain B3728. Codon usage in these two genes, and in another open reading frame (nxi) that was adventitiously isolated during early cloning attempts, shows some characteristic omissions and a strong G + C preference in redundant positions.

Aldose-Ketose Isomerases↗
PubMed1998

Purification of a 58-kDa protein (ER58) from monkey liver microsomes and comparison with protein-disulfide isomerase.

A 58-kDa protein (ER58) was purified from monkey liver to apparent homogeneity. It accounts for more than 3% of microsomal proteins and is highly conserved among several mammalian species. The amino acid compositions of the N-terminal part and that of two internal peptide fragments present strong similarities with the sequence ascribed to phospholipase C-alpha. Numerous proteins exhibiting a high similarity with this sequence have been isolated by other investigators. Their biological function is controversial. Our purified protein is not active as a phosphatidylinositol-specific phospholipase C, protease or carnitine acyl transferase. Although less efficient than authentic protein-disulfide isomerase, ER58 catalyses the glutathione-dependent reduction of insulin and the reorganization of disulfide bonds of randomly oxidized (scrambled) ribonuclease in reducing conditions. In contrast, ER58 is devoid of oxidizing activity on thiol groups of reduced proteins. Many studies suggest that the proteins bearing the phospholipase C-alpha sequence could be considered as protein-disulfide isomerase isozymes. Our results indicate that ER58 is not totally similar to protein-disulfide isomerase in performing thiol :protein-disulfide oxidoreductase reactions and suggest that the two proteins may exert distinct cellular functions.

Amino Acid Sequence↗
PubMed2000

Prolyl isomerases in a minimal cell. Catalysis of protein folding by trigger factor from Mycoplasma genitalium.

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.

Amino Acid Sequence↗
PubMed1990

[Infectious mononucleosis: hemolysis by autoantibodies against triosephosphate isomerase].

Generalized adenopathy and splenomegaly developed in an 18-year-old youth after a severe tonsillitis followed by headache, tiredness and weight loss for several weeks. Infectious mononucleosis (acute Epstein-Barr virus infection) was confirmed by the demonstration of virus-specific antibodies. A reticulocytosis (24%), decreased haptoglobin concentration (0.6 mg/dl) and increased lactate dehydrogenase activity (657 U/l) indicated marked haemolysis. The bone marrow showed increased erythropoiesis with abnormal maturation. Antibodies against triosephosphate isomerase and against blood group marker "i" were demonstrated in the patient's serum. Antibodies against triosephosphate isomerase from the patient's serum were purified by affinity-chromatography. They strongly reacted with the patient's erythrocytes and under complement activation induced an increased 51Cr liberation from marked erythrocytes. No corresponding effect of anti-i-antibodies was noted at 37 degrees C. With the fall in antibodies against triosephosphate isomerase the haemolysis receded and the patient became free of symptoms after 7 weeks.

Adolescent↗
PubMed1977

Effect of polyploidy on phosphoglucose isomerase diversity in Festuca microstachys.

Studies of the inheritance of electrophoretic banding patterns in Festuca microstachys support the hypothesis that three closely related loci, one located in each of the three ancestral genomes, code the multiple phosphoglucose isomerase (glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) variants found in this hexaploid species. The close relationship among the three loci is indicated by the observation that hybrid enzymes of intercistronic origin form when the loci in different genomes carry alleles coding homodimers with unlike migration rates. Homozygous individuals fixed for different alleles in different genomes produce hybrid enzymes and, when self-fertilized, they breed true for isozyme patterns normally found only in the heterozygotes of diploid species. Biochemical diversity due to this "fixed heterozygosity" is high in F. microstachys; although this species is more than 99% self-fertilized the proportion of individuals with at least one heterodimer exceeded 61% in all of the 16 natural populations studied and it exceeded 92% in 11 of the populations. This great biochemical diversity may contribute to the ability of F. microstachys to survive in the wide range of habitats in which it is found over western North America.

Alleles↗
PubMed1991

Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket.

The substrate specificity of thermophilic xylose isomerase from Clostridium thermosulfurogenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and site-directed mutagenesis of the thermophile xylA gene. The orientation of glucose as a substrate in the active site of the thermophilic enzyme was modeled to position the C-6 end of hexose toward His-101 in the substrate-binding pocket. The locations of Met-87, Thr-89, Val-134, and Glu-180, which contact the C-6-OH group of the substrate in the sorbitol-bound xylose isomerase from Arthrobacter [Collyer, C.A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235], are equivalent to those of Trp-139, Thr-141, Val-186, and Glu-232 in the thermophilic enzyme. Replacement of Trp-139 with Phe reduced the Km and enhanced the kcat of the mutant thermophilic enzyme toward glucose, whereas this substitution reversed the effect toward xylose. Replacement of Val-186 with Thr also enhanced the catalytic efficiency of the enzyme toward glucose. Double mutants with replacements Trp-139----Phe/Val-186----Thr and Trp-139----Phe/Val-186----Ser had a higher catalytic efficiency (kcat/Km) for glucose than the wild-type enzyme of 5- and 2-fold, respectively. They also exhibited 1.5- and 3-fold higher catalytic efficiency for D-glucose than for D-xylose, respectively. These results provide evidence that alteration in substrate specificity of factitious thermophilic xylose isomerases can be achieved by designing reduced steric constraints and enhanced hydrogen-bonding capacity for glucose in the substrate-binding pocket of the active site.

Aldose-Ketose Isomerases↗
PubMed1996

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

Bacterial Proteins↗
PubMed1998

Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte.

The enzyme isopentenyl pyrophosphate (IPP) isomerase catalyzes the reversible isomerization of IPP to produce dimethylallyl pyrophosphate, the initial substrate leading to the biosynthesis of carotenoids and many other long-chain isoprenoids. Expression of IPP isomerase, and of two enzymes specific to the carotenoid pathway (lycopene beta-cyclase and beta-carotene-C-4-oxygenase), was followed in the green unicellular alga Haematococcus pluvialis after exposure to high illumination. This alga uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of ketocarotenoids in the cytosol. The carotenoid/chlorophyll ratio increased 3-fold in wild type and 6-fold in a precocious carotenoid-accumulating mutant (Car-3) within 24 h after increasing the illumination from 20 to 150 micromol photon m-2.s-1. Two cDNAs encoding IPP isomerase in Haematococcus, ipiHp1 and ipiHp2, were identified. Although otherwise highly similar (95% identity overall), the predicted sequence of ipiHp1 contained a 12-aa region not found in that of ipiHp2. This was reflected by a size difference between two polypeptides of 34 and 32.5 kDa, both of which reacted with an antibody to the product of ipiHp1. We suggest that the 32.5-kDa form is involved with the carotenoid accumulation in the cytoplasm, since the 32.5-kDa polypeptide was preferentially up-regulated by high light preceding the carotenoid increase and only this form was detected in red cysts.

Amino Acid Sequence↗
PubMed1998

Peroxisomal Delta3-cis-Delta2-trans-enoyl-CoA isomerase encoded by ECI1 is required for growth of the yeast Saccharomyces cerevisiae on unsaturated fatty acids.

We have identified the Saccharomyces cerevisiae gene ECI1 encoding Delta3-cis-Delta2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of (poly)unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32, 000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing Delta3-cis-Delta2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2001

Interaction of the periplasmic peptidylprolyl cis-trans isomerase SurA with model peptides. The N-terminal region of SurA id essential and sufficient for peptide binding.

One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, which is catalyzed by a range of peptidylprolyl cis-trans isomerases. To characterize the interaction between model peptides and the periplasmic peptidylprolyl cis-trans isomerase SurA from E. coli, we employed a chemical cross-linking strategy that has been used previously to elucidate the interaction of substrates with other folding catalysts. The interaction between purified SurA and model peptides was significant in that it showed saturation and was abolished by denaturation of SurA; however the interaction was independent of the presence of proline residues in the model peptides. From results obtained by limited proteolysis we conclude that an N-terminal fragment of SurA, comprising 150 amino acids that do not contain the active sites involved in the peptidylprolyl cis-trans isomerization, is essential for the binding of peptides by SurA. This was confirmed by probing the interaction of the model peptide with the recombinant N-terminal fragment, expressed in Escherichia coli. Hence we propose that, similar to protein disulfide isomerase and other folding catalysts, SurA exhibits a modular architecture composed of a substrate binding domain and distinct catalytically active domains.

Amino Acid Sequence↗
PubMed2001

Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin.

Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.

Catalysis↗