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At least 919 records · Page 51Linked to original sources
PubMed1995

Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-delta 1-dehydrogenase, 3-ketosteroid-delta 5-isomerase and a hypothetical regulatory protein.

The Arthrobacter simplex gene coding for 3-ketosteroid-delta 1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-delta 1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-delta 5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-delta 1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-delta 1-dehydrogenase. The expression of 3-ketosteroid-delta 5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.

Amino Acid Sequence↗
PubMed1985

A fine analysis of glucose-phosphate-isomerase patterns in single preimplantation mouse embryos.

Mouse embryos were derived from eggs heterozygous for alleles of the dimeric enzyme glucose phosphate isomerase (Gpi-1a/Gpi-1b) that had been fertilized with sperm carrying a third allele (Gpi-1c). This particular combination makes it possible to study the activity of the paternally derived as well as the maternally derived genes, the persistence of oocyte-coded enzyme throughout early development and the possible simultaneous expression of both the paternally derived allele and the maternal message. The different isozymes present in single embryos were separated by electrophoresis. The results show that the oocyte-coded glucose phosphate isomerase is gradually replaced by embryo-coded enzyme. Expression of the paternally derived allele was first detected at the morula stage, during which the translation of the maternally derived message seemed to be either exhausted or below the detection limit of our system. Some oocyte-coded enzyme persisted until after implantation.

Animals↗
PubMed1993

The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant.

Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.

Amino Acid Sequence↗
PubMed2004

The crystal structure of glucose-6-phosphate isomerase from Leishmania mexicana reveals novel active site features.

Glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate in glycolysis and gluconeogenesis, and in the recycling of hexose-6-phosphate in the pentose phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order Kinetoplastida are important human parasites responsible for African sleeping sickness, Chagas' disease and leishmaniases, respectively. In these parasites, glycolysis is an important (and in some cases the only) metabolic pathway for ATP supply. The first seven of the 10 enzymes that participate in glycolysis, as well as an important fraction of the enzymes of the pentose phosphate pathway, are compartmentalized in peroxisome-like organelles called glycosomes. The dependence of the parasites on glycolysis, the importance of the pentose phosphate pathway in defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes contained in the glycosome as potential targets for drug design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana) glucose-6-phosphate isomerase. A comparison of the atomic structure of L. mexicana, human and other mammalian PGIs, which highlights unique features of the parasite's enzyme, is presented.

Amino Acid Sequence↗
PubMed1994

Investigation of the role of sterol delta 8-->7-isomerase in the sensitivity of Saccharomyces cerevisiae to fenpropimorph.

Treatment of Saccharomyces cerevisiae with the morpholine fungicide fenpropimorph was examined using both a wild-type and a mutant strain (erg2) defective in sterol delta 8-->7-isomerase. No resistance to fenpropimorph was observed in the mutant strain after 3 days, although after 7 days the mutant and the wild-type strains had grown in concentrations of fenpropimorph close to the saturating dose. Re-inoculation of both strains into fresh medium containing fenpropimorph resulted in continued growth and this adaptation to fungicide tolerance was lost on subculture in the absence of fenpropimorph. Analysis of the sterols present in the cells indicated that fenpropimorph treatment resulted in the accumulation of delta 8,14-sterols. This accumulation and the corresponding depletion of ergosterol were correlated with growth inhibition rather than the presence of delta 8-sterols. Together with an absence of gene dosage effect for ERG2 on fenpropimorph sensitivity, this supports the hypothesis that sterol delta 8-->7-isomerase inhibition does not contribute to the fungicidal activity of fenpropimorph.

Base Sequence↗
PubMed1996

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli.

A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the D-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M(r) 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli.

Aldose-Ketose Isomerases↗
PubMed1997

Histidine 109 in peptidyl-prolyl cis-trans isomerase of Bacillus subtilis plays an important role in catalysis and in cyclosporin A binding.

The cyclophilin of Bacillus subtilis has a moderate affinity to cyclosporin A (IC50: 120 nM) and low catalytic activity (Kcat/ Km: 1.1 microM-1 s-1) when compared to other ubiquitous peptidyl-prolyl cis-trans isomerases (PPIases). The active site residues V52, H90 and H109, which are not conserved within other peptidyl-prolyl cis-trans isomerases, were found to play an important role in cyclosporin A binding and catalytic activity. In this work we report on double mutations of these residues, which greatly improved cyclosporin A affinity and catalytic activity. The H90N/H109W mutation displayed an IC50 value of 46 nM whereas the V52M/H109F mutation exhibited over 18-fold higher catalytic activity than that detected for wild-type PPIase. The mutations H109W and H109F of the B. subtilis PPIase showed no change in cyclosporin A affinity and catalytic activity between pH 6 and 8. In contrast, wild-type PPIase (H109) showed up to 10-fold reduction below pH 7.5, both in cyclosporin A affinity and in catalytic activity. These findings clearly underline the importance of the unique H109 residue in the B. subtilis enzyme.

Bacillus subtilis↗
PubMed2002

Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to D-tagatose using the enzyme.

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.

Aldose-Ketose Isomerases↗
PubMed2005

Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus.

Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by site-directed mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and/or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni2+ saturating conditions. Further, the Ni2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.

Amino Acid Substitution↗
PubMed2006

Protein disulfide isomerase family proteins involved in soybean protein biogenesis.

Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a', and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.

Amino Acid Sequence↗
PubMed1973

Chromosome assignments in man of the genes for two hexosephosphate isomerases.

Thirty-seven clones of somatic cell hybrids between human and mouse cells were examined for retention of human chromosomes and expression of human constitutive enzymes. Human glucosephosphate isomerase and chromosome F-19 were retained or lost concordantly, as were human mannosephosphate isomerase and chromosome C-7. The genes for the enzymes are thus assigned to these two chromosomes.

Animals↗
PubMed1983

Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning.

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIII restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose.

Aldose-Ketose Isomerases↗
PubMed1984

Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli.

A 4.2-kilobase-pair fragment of the Escherichia coli chromosome which contains the genes for xylose isomerase and xylulose kinase was cloned into plasmid pBR322. The hybrid plasmid (designated pECX14) complements strains deficient in either or both of the two enzymes. Deletion derivatives of pECX14 were used to localize the two genes on the cloned fragment. The entire nucleotide sequence of the cloned fragment was determined. Open reading frames which, if translated, would encode proteins of molecular weights 54,000 and 52,000 were found. These were identified as the isomerase and kinase structural genes, respectively.

Aldose-Ketose Isomerases↗
PubMed1987

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.

Aldose-Ketose Isomerases↗
PubMed1996

Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase.

The Thermus thermophilus xylA gene encoding xylose (glucose) isomerase was cloned and expressed in Saccharomyces cerevisiae under the control of the yeast PGK1 promoter. The recombinant xylose isomerase showed the highest activity at 85 degrees C with a specific activity of 1.0 U mg-1. A new functional metabolic pathway in S. cerevisiae with ethanol formation during oxygen-limited xylose fermentation was demonstrated. Xylitol and acetic acid were also formed during the fermentation.

Aldose-Ketose Isomerases↗
PubMed2000

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

Alkyl and Aryl Transferases↗
PubMed1971

Free and immobilized glucose isomerase from Streptomyces phaeochromogenes.

Properties were determined of the glucose isomerase from Streptomyces phaeochromogenes NRRL B-3559. The enzyme exhibited a temperature optimum of 80 C and a pH optimum of about 8. The effect of various buffers on activity of the enzyme and the optimum pH were studied. Michaelis constants for glucose and Mg(2+) were 0.25 and 0.025 m, respectively. Co(2+) enhanced enzyme activity. A functional polyacrylamide-entrapped glucose isomerase was prepared. The conditions for entrapment and use of the bound enzyme were examined.

Acrylates↗