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PubMed2001

Cloning and characterization of a novel gene encoding L-ribose isomerase from Acinetobacter sp. strain DL-28 in Escherichia coli.

The gene encoding a novel L-ribose isomerase (L-RI) from Acinetobacter sp. was cloned into Escherichia coli and nucleotide sequence was determined. The gene corresponded to an open reading frame of 747 bp that codes for a deduced protein of 249 amino acids, which showed no amino acid sequence similarity with any other sugar isomerases. After expression of the gene in E. coli using pUC118 the recombinant L-RI was purified to homogeneity using different chromatographic methods. The overall enzymatic properties of the purified recombinant L-RI were the same as those of the authentic L-RI. To our knowledge, this is the first time report concerning the L-RI gene.

Acinetobacter↗

PubMed2001

Studies on the function of yeast protein disulfide isomerase in renaturation of proteins.

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.

Binding Sites↗

PubMed1999

Biochemical evidence that Escherichia coli hyi (orf b0508, gip) gene encodes hydroxypyruvate isomerase.

We found a significant activity of hydroxypyruvate isomerase in Escherichia coli clone cells harboring an E. coli gene (called orf b0508 or gip), which is located downstream of the glyoxylate carboligase gene. We newly designated the gene hyi. The enzyme was purified from cell extracts of the E. coli clone. The enzyme had a molecular mass of 58 kDa and was composed of two identical subunits. The optimum pH for the isomerization of hydroxypyruvate was 6.8-7.2. The enzyme required no cofactor. It exclusively catalyzed the isomerization between hydroxypyruvate and tartronate semialdehyde. The apparent K(m) value for hydroxypyruvate was 12.5 mM. The amino acid sequence of E. coli hydroxypyruvate isomerase is highly similar to those of glyoxylate-induced proteins, Gip, found widely from prokaryotes to eukaryotes.

Aldose-Ketose Isomerases↗

PubMed2003

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.

Cell Division↗

PubMed1999

Purification and properties of phosphoglucose isomerases of Trypanosoma cruzi.

Glucosephosphate isomerase (PGI; EC 5.3.1.9) of Trypanosoma cruzi epimastigotes was found in about the same proportion in the glycosome and the cytosol. This subcellular distribution is similar to that of Leishmania mexicana, but contrasts with that of T. brucei bloodstream form, where the enzyme is essentially restricted to the glycosome. Glucosephosphate isomerase was highly purified from a glycosome-enriched fraction and to about 70% purity from the soluble extract. Both enzymes displayed Michaelis-Menten-Henri kinetics. Km values for fructose 6-phosphate were 0.125 +/- 0.07 and 0.80 +/- 0.10 mM for the glycosomal and the cytosolic PGIs, respectively. Erythrose-4-phosphate, 6-phosphogluconate and mannose-6-phosphate were inhibitors for both PGIs. Phosphogluconate and erythrose phosphate showed higher affinity for cytosolic PGI than for glycosomal PGI, by 2.5- and 4-fold respectively. The PGIs differed slightly in their isoelectric point (7.1 +/- 0.15 and 7.5 +/- 0.12) and optimum pH range. Both PGIs also differed in their chromatographic properties (ion-exchange and phenyl Sepharose), indicating a difference in charge and hydrophobicity, with the glycosomal enzyme being more hydrophobic. The molecular mass of both PGIs was 186,000 +/- 9000 Da, which is higher than that of other known PGIs, including those from T. brucei and other trypanosomatids. The molecular mass of the subunit, 63 kDa, is similar to that of PGIs from other sources. It appears that PGIs from T. cruzi are trimeric, in contrast with all other known PGIs which are dimeric.

Animals↗

PubMed2004

Chronic axonal neuropathy with triosephosphate isomerase deficiency.

A patient with triosephosphate isomerase deficiency resulting from compound heterozygote mutation is described. Chronic axonal neuropathy was identified on clinical and neurophysiologic grounds and confirmed by sural nerve biopsy. This report describes the first biopsy-proven case confirming that peripheral neuropathy can occur with triosephosphate isomerase deficiency.

Axons↗

PubMed2001

Structure and enzymology of Delta5-3-ketosteroid isomerase.

The three-dimensional structures of Delta5-3-ketosteroid isomerases from two different bacterial species have been determined. The structures reveal an unusually apolar active site, in which each of several competitive inhibitors of the enzyme are held by two hydrogen bonds with the general acids Tyr14 and Asp99, and by hydrophobic interactions. The hydrogen bond between the Tyr14 hydroxyl and the C3 oxyanion of a transition-state analog is a low-barrier hydrogen bond, as indicated by a highly deshielded nuclear magnetic resonance. Structural and other biochemical studies have enabled the proposal of a detailed catalytic mechanism for Delta5-3-ketosteroid isomerase and provided a major thrust towards understanding the mechanism not only in chemical terms but also in energetics terms.

Binding Sites↗

PubMed1998

Synthesis and evaluation of a new inhibitor of phosphoglucose isomerases: the enediolate analogue 5-phospho-D-arabinohydroxamate.

Designed as a high energy intermediate analogue inhibitor of the potent chemotherapeutic target phosphoglucose isomerases, 5-phospho-D-arabinohydroxamate was efficiently synthesized in a two steps procedure. To date, it proved to be the strongest competitive inhibitor with respect to substrate D-fructose-6-phosphate (Ki down to 98 nM and Km/Ki values up to 513). A comparative inhibition study of this compound and other known strong inhibitors on phosphoglucose isomerases from three different sources is also reported.

Animals↗

PubMed2002

A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure.

A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.

Aldose-Ketose Isomerases↗

PubMed1999

Unusual stability of a multiply nicked form of Plasmodium falciparum triosephosphate isomerase.

BACKGROUND: The limited proteolytic cleavage of proteins can result in distinct polypeptides that remain noncovalently associated so that the structural and biochemical properties of the 'nicked' protein are virtually indistinguishable from those of the native protein. The remarkable observation that rabbit muscle triosephosphate isomerase (TIM) can be multiply nicked by subtilisin and efficiently religated in the presence of an organic solvent formed the stimulus for our study on a homologous system, Plasmodium falciparum triosephosphate isomerase (PfTIM). RESULTS: The subtilisin nicked form of PfTIM was prepared by limited proteolysis using subtilisin and the major fragments identified using electrospray ionization mass spectrometry. The order of susceptibility of the peptide bonds in the protein was also determined. The structure of the nicked form of TIM was investigated using circular dichroism, fluorescence and gel filtration. The nicked enzyme exhibited remarkable stability to denaturants, although significant differences were observed with the wild-type enzyme. Efficient religation could be achieved by addition of an organic cosolvent, such as acetonitrile, in the presence of subtilisin. Religation was also demonstrated after dissociation of the proteolytic fragments in guanidinium chloride, followed by reassembly after removal of the denaturant. CONCLUSIONS: The eight-stranded beta8/alpha8 barrel is a robust, widely used protein structural motif. This study demonstrates that the TIM barrel can withstand several nicks in the polypeptide backbone with a limited effect on its structure and stability.

Amino Acid Sequence↗

PubMedDate not supplied

Death of mouse embryos that lack a functional gene for glucose phosphate isomerase.

A null allele of the Gpi-1s structural gene, that encodes glucose phosphate isomerase (GPI-1; E.C. 5.3.1.9), arose in a mutation experiment and was designated Gpi-1sa-m1H. The viability of homozygotes has been investigated. No offspring homozygous for the null allele were produced by intercrossing two heterozygotes, so the homozygous condition was presumed to be embryonic lethal. Embryos were produced by crossing Gpi-1sa/null heterozygous females and Gpi-1sb/null heterozygous males. Homozygous null embryos were identified at different stages of development by electrophoresis and staining either for GPI-1 alone or GPI-1 plus phosphoglycerate kinase (PGK) activity. At 6 1/2 and 7 1/2 days post coitum homozygous null embryos were present at approximately the expected 25% frequency (37/165; 22.4% overall) although at 7 1/2 days the homozygous null embryos tended to be small. By 8 1/2 days most homozygous null embryos were developmentally retarded and had not developed significantly further than at 7 1/2 days; some were dead or dying. By 9 1/2 days the homozygous null conceptus was characterised by a small implantation site that contained trophoblast and often a small amount of extraembryonic membrane. Surviving trophoblast tissue was also detectable at 10 1/2 days. Previous studies have shown that oocyte-coded GPI-1 persists only until 5 1/2 or 6 1/2 days. Survival of homozygous null embryos to 7 1/2 or 8 1/2 days and survival of certain extraembryonic tissue to 10 1/2 days suggests that the homozygous null condition may not be cell-lethal although it is certainly embryo-lethal. Mutant cells that are deficient in glycolysis may use the pentose phosphate shunt to bypass the block in glycolysis created by the deficiency of glucose phosphate isomerase, and/or might be rescued by the transport, from the maternal blood, of energy sources other than glucose (such as glutamine). Either strategy may only permit slow cell growth that would not be adequate to support normal embryogenesis. Transport of maternal nutrients would be more efficient to the trophoblast and extraembryonic membranes and this may help to explain why these tissues survive for longer than the embryo itself. The morphological similarity between homozygous nulls and androgenetic conceptuses, where the trophoblast also survives better than the embryo, is discussed.

Animals↗

PubMed1995

Enzymatic and nonenzymatic polarizations of alpha,beta-unsaturated ketosteroids and phenolic steroids. Implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5-3-ketosteroid isomerase.

Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

PubMed1995

Ultraviolet resonance Raman spectroscopy of delta 5-3-ketosteroid isomerase revisited: substrate polarization by active-site residues.

The delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes extremely rapid conversion of delta 5- to delta 4-3-ketosteroids by a conservative intramolecular proton transfer via an enolic intermediate. The competitive inhibitor 19-nortestosterone displays marked spectroscopic changes upon binding to the enzyme, but the mechanisms responsible for these changes have not been unequivocally established. Ultraviolet resonance Raman (UVRR) spectra are reported for 19-nortestosterone in acid solutions and for this ligand when bound to delta 5-3-ketosteroid isomerase, as well as to its D38N and Y14F/D38N mutants. The frequencies of UVRR bands associated with C = O and C = C stretching can be used to monitor the state of polarization of the enone fragment of the steroid and the effects of the catalytic side chains, Tyr-14 and Asp-38, on these polarizations. Strong polarization is indicated by marked frequency downshifts of the C = O and C = C bands in the native protein; the downshifts are diminished by the mutations of these catalytic residues. The lower polarizing effects of the Y14F and D38N single mutants and the Y14F/D38N double mutant indicate that most of the polarization of the conjugated ketone is attributable to hydrogen-bond donation by the hydroxyl group of Tyr-14. A smaller contribution of Asp-38 is detected which is, in part, cooperative with that of Tyr-14. Reference spectra of hydrogen-bonded and protonated forms of 19-nortestosterone are reassigned, on the basis of the species identification of D. C. Hawkinson and R. M. Pollack [(1993) Biochemistry 32, 694-698].(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1995

Complementation of fragments of triosephosphate isomerase defined by exon boundaries.

Chicken triosephosphate isomerase (TIM) has been fragmented by inserting single "splits" at three separate exon/exon boundaries, and the complexes have been assayed for catalytic activity. In vivo studies show that the expression of both portions of each of the three different split genes complements the TIM deficiency of Escherichia coli strain DF502 when grown on selective media. The expression of only one fragment of each split gene does not complement the TIM-minus genotype. To assess the catalytic activity that derives from the fragmented protein complex, the individual peptide products of one of the three split genes were expressed and purified. The purified complex showed isomerase activity, albeit of low specific catalytic activity. A catalytically active multichain complex composed of separate peptide products of a gene singly split at exon/exon junctions has thus been created.

Animals↗

PubMed1991

Energetics of 3-oxo-delta 5-steroid isomerase: source of the catalytic power of the enzyme.

Knowledge of the partitioning of the putative dienol intermediate (2) by steroid isomerase (KSI) (Hawkinson et al. 1991), in conjunction with various steady-state kinetic parameters, allows elucidation of the detailed free energy profile for the KSI-catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3). This free energy profile shows four kinetically significant energy barriers (substrate binding, the two chemical steps, and dissociation of product) that must be traversed upon conversion of 1 to 3. Thus, no single step of the catalytic cycle is cleanly rate-limiting. The source of the catalytic power of KSI is discussed via comparison of the free energy profile for the KSI-catalyzed isomerization with those for the acetate-catalyzed isomerization and the aqueous reaction at pH 7. Similarities between the energetics of the KSI-catalyzed and triosephosphate isomerase catalyzed reactions are also noted.

Androstenedione↗

PubMed1994

Role of the divalent metal ion in sugar binding, ring opening, and isomerization by D-xylose isomerase: replacement of a catalytic metal by an amino acid.

The distinct roles of the two magnesium ions essential to the activity of D-xylose isomerase from Streptomyces olivochromogenes were examined. The enzyme-magnesium complex was isolated, and the stoichiometry of cation binding determined by neutron activation analysis to be 2 mol of magnesium per mole of enzyme. A plot of Mg2+ added versus Mg2+ bound to enzyme is consistent with apparent KD values of < or = 0.5-1.0 mM for one Mg2+ and < or = 2-5 mM for the second. A site-directed mutant of D-xylose isomerase was designed to remove the tighter, tetracoordinated magnesium binding site (site 1, Mg-1); Glu180 was replaced with Lys180. The stoichiometry of metal binding to this mutant, E180K, is 1 mol of magnesium per mole of enzyme. Ring-opening assays with 1-thioglucose (H2S released upon ring opening) show E180K catalyzes the opening of the sugar ring at 20% the rate of the wild-type, but E180K does not catalyze isomerization of glucose to fructose. Thus, the magnesium bound to Glu180 is essential for isomerization but not essential for ring opening. The X-ray crystallographic structures of E180K in the absence of magnesium and in the presence and absence of 250 mM glucose were obtained to 1.8-A resolution and refined to R factors of 17.7% and 19.7%, respectively. The wild-type and both E180K structures show no significant structural differences, except the epsilon-amino group of Lys180, which occupies the position usually occupied by the Mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗

PubMed1994

Mechanism of the reaction catalyzed by delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: kinetic properties of a modified enzyme in which tyrosine 14 is replaced by 3-fluorotyrosine.

Tyrosine 14 of delta 5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since its replacement by phenylalanine results in a decrease in kcat by a factor of 10(-4.7). This result and the fact that this residue resides in the enzyme's substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate. In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKa' of the phenol hydroxyl should be decreased by about 1.5 log units. Site specificity of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine. We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in kcat. We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like. An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data.

Base Sequence↗

PubMed2001

Crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho-D-arabinonate identifies the role of Glu357 in catalysis.

Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) catalyzes the second step in glycolysis, the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. We determined the X-ray crystal structure of rabbit PGI complexed with a competitive inhibitor of isomerase activity, 5-phospho-D-arabinonate (5PAA), at 1.9 A resolution. 5PAA is a better mimic of the proposed cis-enediol(ate) intermediate than 6-phospho-D-gluconate, which was used in a previously reported crystal structure of rabbit PGI. The orientation of 5PAA bound in the enzyme active site predicts that active site residue Glu357 is the residue that transfers a proton between C2 and C1 of the proposed cis-enediol(ate) intermediate. Amino acid residues Arg272 and Lys210 are predicted to be involved in stabilizing the negative charge of the intermediate.

Animals↗