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At least 775 records · Page 43Linked to original sources
PubMed2004

The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis.

Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site. This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea. Whilst the mechanism of the latter family is thought to proceed through a cis-enediol intermediate, analysis of the structure of PfPGI in the presence of inhibitors has led to the suggestion that the mechanism of this enzyme involves the metal-dependent direct transfer of a hydride between C1 and C2 atoms of the substrate. To gain further insight in the reaction mechanism of PfPGI, the structures of the free enzyme and the complexes with the inhibitor, 5-phospho-d-arabinonate (5PAA) in the presence and absence of metal have been determined. Comparison of these structures with those of equivalent complexes of the eukaryotic PGIs reveals similarities at the active site in the disposition of possible catalytic residues. These include the presence of a glutamic acid residue, Glu97 in PfPGI, which occupies the same position relative to the inhibitor as that of the glutamate that is thought to function as the catalytic base in the eukaryal-type PGIs. These similarities suggest that aspects of the catalytic mechanisms of these two structurally unrelated PGIs may be similar and based on an enediol intermediate.

Archaeal Proteins↗
PubMed2004

Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme.

Recent studies have implied that the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid. PhIPMI is thus considered a key to understanding the fundamental metabolism of the earliest organisms. We describe for the first time the crystal structure of PhIPMI-s, which displays dual substrate specificity. The crystal structure unexpectedly shows that four molecules create an interlocked assembly with intermolecular disulfide linkages having a skewed 222 point-group symmetry. Although the overall fold of the PhIPMI-s monomer is related closely to domain 4 of the aconitase (ACN), one alpha-helix in the ACN structure is replaced by a short loop with relatively high temperature factor values. Because this region is essential for discriminating the structurally similar substrate based on interactions with its diversified gamma-moiety, the loop structure in the PhIPMI-s must be dependent on the presence of a substrate. The flexibility of the loop region might be a structural basis for recognizing both hydrophobic and hydrophilic gamma-moieties of two distinct substrates, isopropylmalate and homocitrate.

Amino Acid Sequence↗
PubMed2005

Diversification of catalytic activities and ligand interactions in the protein fold shared by the sugar isomerases, eIF2B, DeoR transcription factors, acyl-CoA transferases and methenyltetrahydrofolate synthetase.

Evolution of diverse catalytic and ligand-binding activities in a given protein fold is a widely observed phenomenon in the protein-domain universe. However, the details of this evolutionary process, general principles, if any, and implications for origins of particular catalytic mechanisms are poorly understood in many common protein folds. Taking advantage of the wealth of currently available protein structure and sequence data, we explore these issues in the context of a large assemblage of biochemically diverse protein domains sharing a common origin, namely the sugar isomerases, translation factor eIF2B, ligand-binding domains of the DeoR-family transcription factors, acetyl-CoA transferases and methenyltetrahydrofolate synthetase. We show that in at least three independent instances, including the sugar-binding domains of the DeoR family transcription factors, this domain has been used as small molecule sensor coupled to helix-turn-helix DNA-binding domains. In at least two of these instances the domain functions as a non-catalytic sensor of ligands. We provide evidence that the ancestral version of this fold was a distinct version of the Rosmann-like folds, which probably possessed two distinct ligand-binding areas that were differentially utilized in different descendents. Analyzing the sequences and structures of proteins in this fold we show that there are two principal factors related to the origin of catalytic diversity in this fold. Firstly, specific inserts and extension added to the core domain on multiple occasions in evolution have affected the access to the active site regions, and thereby allowed for different substrates and allosteric regulators. The second major factor appears to be the emergence of considerable diversity of family-specific residues with important biochemical roles. Interestingly, proteins of this fold, which catalyze similar reactions on similar substrates, might possess very distinctive sets of active residues required for substrate binding catalysis. In particular, different sugar isomerases or acyl transferases in this fold might show distinct constellations of active site residues. These findings suggest that whereas ligand-binding, and even generic catalytic ability emerged early in the evolution of the fold, the specific catalytic mechanisms appear to have independently emerged on multiple occasions in the generic precursors of this fold.

Aldose-Ketose Isomerases↗
PubMed2006

Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production.

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 A resolution. The subunit structure of ECAI is organised into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.

Aldose-Ketose Isomerases↗
PubMed2006

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity.

Pseudomonas stutzeri L-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with L-rhamnose and D-allose which has different configurations of C4 and C5 from L-rhamnose, were determined at a resolution of 2.0 A, 1.97 A, and 1.97 A, respectively. P. stutzeri L-RhI has a large domain with a (beta/alpha)(8) barrel fold and an additional small domain composed of seven alpha-helices, forming a homo tetramer, as found in E. coli L-RhI and D-xylose isomerases (D-XIs) from various microorganisms. The beta1-alpha1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding beta1-alpha1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with L-rhamnose and D-allose show that both substrates are nicely fitted to the substrate-binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the beta1-alpha1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of L-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.

Aldose-Ketose Isomerases↗
PubMed2004

A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase.

Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase.

Aldose-Ketose Isomerases↗
PubMed2005

Increase of soluble expression in Escherichia coli cytoplasm by a protein disulfide isomerase gene fusion system.

Human protein disulfide isomerase (PDI) was selected as a fusion partner to construct a gene expression system to enhance the solubility of recombinant protein in Escherichia coli. DREBIII-1, a plant specific transcriptional factor, was found to mainly form inclusion bodies when expressed in either His-tagged or GST-fusion systems in E. coli. In contrast, when fused with PDI, the expressed DREBIII-1 was in a highly soluble and biologically active form. Two fusion proteins, HDP and HPD, were generated by positioning DREBIII-1 at the N-terminal and C-terminal of PDI, respectively. After purification, HDP exhibited a higher stability and showed only one band on SDS-PAGE, while HPD degraded as several bands. HDP was verified to have the biological function of PDI by isomerase activity assay; meanwhile, it also presented the DNA binding and transcriptional activation characteristic of DREBIII-1 in fluorescence quenching and yeast one-hybrid experiments. The PDI fusion expression system was demonstrated to be highly efficient in generating not only soluble but functional desired proteins.

Brassica napus↗
PubMed2006

Crystal structure of the bb' domains of the protein disulfide isomerase ERp57.

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.

Amino Acid Sequence↗
PubMed1990

Phosphorus-31 nuclear magnetic resonance spectroscopy reveals two conformational forms of chloroacetol phosphate-bound triosephosphate isomerase.

Chloroacetol phosphate covalently reacts with Glu-165 in the catalytic center of triosephosphate isomerase. Reaction of the enzyme with the substrate analogue results in two 31P resonances at 6.8 and 5.5 ppm. Dissociation with guanidinium chloride results in a single resonance at 4.5 ppm. Reassociation and redimerization of the triosephosphate isomerase-chloroacetol phosphate complex restores only the resonance at 5.5 ppm. The two 31P resonances appear to represent different conformations of the enzyme which are trapped upon reaction with the affinity label.

Affinity Labels↗
PubMed1998

The loop opening/closing motion of the enzyme triosephosphate isomerase.

To explore the origin of the large-scale motion of triosephosphate isomerase's flexible loop (residues 166 to 176) at the active site, several simulation protocols are employed both for the free enzyme in vacuo and for the free enzyme with some solvent modeling: high-temperature Langevin dynamics simulations, sampling by a "dynamics driver" approach, and potential-energy surface calculations. Our focus is on obtaining the energy barrier to the enzyme's motion and establishing the nature of the loop movement. Previous calculations did not determine this energy barrier and the effect of solvent on the barrier. High-temperature molecular dynamics simulations and crystallographic studies have suggested a rigid-body motion with two hinges located at both ends of the loop; Brownian dynamics simulations at room temperature pointed to a very flexible behavior. The present simulations and analyses reveal that although solute/solvent hydrogen bonds play a crucial role in lowering the energy along the pathway, there still remains a high activation barrier. This finding clearly indicates that, if the loop opens and closes in the absence of a substrate at standard conditions (e.g., room temperature, appropriate concentration of isomerase), the time scale for transition is not in the nanosecond but rather the microsecond range. Our results also indicate that in the context of spontaneous opening in the free enzyme, the motion is of rigid-body type and that the specific interaction between residues Ala176 and Tyr208 plays a crucial role in the loop opening/closing mechanism.

Alanine↗
PubMed2001

Functions of the conserved anionic amino acids and those interacting with the substrate phosphate group of phosphoglucose isomerase.

Phosphoglucose isomerase catalyzes the isomerization between glucose 6-phosphate and fructose 6-phosphate in cytoplasm, and functions as autocrine motility factor and neuroleukin outside the cells. A phosphoglucose isomerase from Bacillus stearothermophilus (pgiA) was subjected to mutagenesis study to address the catalytic function of the conserved anionic residues and those probably interacting with the phosphate group of substrates. The results suggest that Glu290 works concertedly with His311 as a general acid-base pair to initiate the isomerization step, and Glu150 assists the base function of His311. The conserved loop structure consisting of Gly205-Gly206-Arg207 plays a critical role for the recognition of substrates.

Amino Acid Substitution↗
PubMed2002

Species specificity of the cytokine function of phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) is a cytosolic glycolytic enzyme that also functions as an extracellular cytokine (neuroleukin/autocrine motility factor (AMF)/maturation factor). Contrary to mammalian PGI, bacterial PGI was not internalized by the PGI/AMF receptor (gp78/AMF-R) and neither bacterial nor yeast PGI competed with mammalian PGI for receptor binding and internalization. Furthermore, while the bacterial, yeast and mammalian preparations all exhibited isomerase activity, only mammalian PGI stimulated the motility of NIH-3T3 fibroblasts. The conserved active site of PGI is therefore not sufficient for receptor binding and cytokine activity of PGI. However, synthetic peptides corresponding to distinct peripheral mammalian PGI sequences did not inhibit internalization of mammalian PGI. Our data therefore argue that the cytokine activity of PGI is specific to mammalian PGI but cannot exclude the possibility that the receptor binding motif of PGI is complex and includes elements within and without the active site.

3T3 Cells↗
PubMed1998

Study on the interactions between protein disulfide isomerase and target proteins, using immobilization on solid support.

Interaction between protein disulfide isomerase, possessing not only isomerase but also chaperone-like activity, and olygomeric enzyme, GAPDH, has been studied using technique of immobilization on insoluble support. PDI dimers bound to CNBr-activated Sepharose were shown to possess high TPOR activity as well as the ability to reactivate lysozyme. Immobilized PDI was not found to interact neither with soluble tetrameric GAPDH, nor with soluble denatured GAPDH. However, soluble PDI binds effectively to immobilized GAPDH monomers; Kd was found to be 3.7 x 10(-6) M, stoichiometry 0.824 mole PDI monomers per mole GAPDH monomers. Immobilized GAPDH tetramers do not interact with PDI. These observations are also confirmed by the data on electrophoresis of proteins bound to immobilized GAPDH monomers and tetramers. The ability of PDI to interact with denatured protein form, but not with the native one, is considered to be evidence of chaperone-like activity of the enzyme.

Disulfides↗
PubMed1999

R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays.

Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay.

Arginine↗
PubMed2000

Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants.

Protein disulfide isomerase (PDI) exhibits both an oxido-reductase and an isomerase activity on proteins containing cysteine residues. These activities arise from two active sites, both of which contain pairs of redox active cysteines. We have developed two simple in vivo assays for these activities of PDI, based on the demonstration that PDI can complement both a dsbA mutation and a dsbC mutation when expressed to the periplasm of Escherichia coli. We constructed a variety of mutants in and around the active sites of PDI and analysed them using these complementation assays. Our analysis showed that the active site amino acid residues have a major role in determining the activities exhibited by PDI, particularly the N-terminal cysteine of the N-terminal active site. The roles of the histidine residue at position 38 and the glutamic acid residue at position 30 were also studied using these assays. The results show that these two in vivo assays should be useful for rapid screening of mutants in PDI prior to purification and detailed biochemical analysis.

Amino Acid Sequence↗
PubMed2000

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium.

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.

Amino Acid Sequence↗
PubMed1998

Carbohydrate-deficient glycoprotein syndrome type 1 with profound thrombocytopenia and normal phosphomannomutase and phosphomannose isomerase activities.

We report siblings with a variant of carbohydrate-deficient glycoprotein syndrome, type 1 (CDGS1), characterized by normal phosphomannomutase and phosphomannose isomerase activities, severe thrombocytopenia, and respiratory compromise. Each infant died after a course of intensive care, suggesting that infants with CDGS1 and normal phosphomannomutase and phosphomannose isomerase activities may have a more severe CDGS1 phenotype.

Congenital Disorders of Glycosylation↗
PubMed1999

Structure determination and refinement of the Al3+ complex of the D254,256E mutant of Arthrobacter D-xylose isomerase at 2.40 A resolution. Further evidence for inhibitor-induced metal ion movement.

The structure of the D254.256E double mutant of Arthrobacter xylose isomerase with Al3+ at both metal-binding sites was determined by the molecular replacement method at a conventional R-factor of 0.179. Binding of the two Al3+ does not alter the overall structure significantly. However, there are local rearrangements in the octahedral co-ordination sphere of the Al3+. The inhibitor molecule moves somewhat away from the active site. Furthermore, evidence was revealed for metal ion movement from site 2(1) to site 2(2) upon double mutation. Xylose isomerase requires two divalent metal cations for activation. The catalytic metal ion is translocated 1.8 A away from its initial position during the catalytic reaction. The fact that both activating and inactivating metals (including Al3+) were found exclusively at a single location in the double mutant was an indication that the consequently missing shuttle may account for the crippled catalytic efficiency.

Aldose-Ketose Isomerases↗