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Sample records for galactokinase

  1. Galactokinase promiscuity: a question of flexibility?

    PubMed

    McAuley, Megan; Kristiansson, Helena; Huang, Meilan; Pey, Angel L; Timson, David J

    2016-02-01

    Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering. PMID:26862196

  2. Galactokinase activity in Streptococcus thermophilus

    SciTech Connect

    Hutkins, R.; Morris, H.A.; McKay, L.L.

    1985-10-01

    ATP-dependent phosphorylation of (/sup 14/C)galactose by 11 strains of streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal/sup +/) strains than in galactose-nonfermenting (Gal/sup -/) strains. Lactose-grown, Gal/sup -/ cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal/sup +/ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted, growth on galactose commenced, and galK increased from 0.025 to 0.22 ..mu..mol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal/sup +/ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal/sup -/ strains.

  3. Structure activity relationships of human galactokinase inhibitors.

    PubMed

    Liu, Li; Tang, Manshu; Walsh, Martin J; Brimacombe, Kyle R; Pragani, Rajan; Tanega, Cordelle; Rohde, Jason M; Baker, Heather L; Fernandez, Elizabeth; Blackman, Burchelle; Bougie, James M; Leister, William H; Auld, Douglas S; Shen, Min; Lai, Kent; Boxer, Matthew B

    2015-02-01

    Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor. PMID:25553891

  4. Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

    PubMed Central

    Lee, R T; Peterson, C L; Calman, A F; Herskowitz, I; O'Donnell, J J

    1992-01-01

    A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationship between the disease locus for galactokinase deficiency galactosemia, which is responsible for cataracts in newborns and possibly presenile cataracts in adults, and the two galactokinase loci is unknown. Images PMID:1438294

  5. Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization.

    PubMed

    Lobo-Rojas, Ángel E; González-Marcano, Eglys B; Valera-Vera, Edward A; Acosta, Héctor R; Quiñones, Wilfredo A; Burchmore, Richard J S; Concepción, Juan L; Cáceres, Ana J

    2016-10-01

    Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9kDa and 51.3kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108mM (TcGALK-1) and 0.091mM (TcGALK-2) and for ATP 0.36mM (TcGALK-1) and 0.1mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source. PMID:27312997

  6. pH-Rate Profiles Support a General Base Mechanism for Galactokinase (Lactococcus lactis)

    PubMed Central

    Reinhardt, Laurie A.; Thoden, James B.; Peters, Greg S.; Holden, Hazel M.; Cleland, W.W.

    2013-01-01

    Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-D-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10,000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH–kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation. PMID:23872454

  7. Ultra Fast and Sensitive Liquid Chromatography Tandem Mass Spectrometry Based Assay for Galactose-1-Phosphate Uridylyltransferase and Galactokinase Deficiencies

    PubMed Central

    Li, Yijun; Ptolemy, Adam S.; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T.

    2013-01-01

    The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([13C6]-uridine diphosphate galactose in GALT assay and [13C6]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4 ± 4.2 and GALK activity of 1.8 ± 0.47 (mean ± SD) µmol·(g Hgb) −1·hr−1. Erythrocyte GALT activities in a cohort of 16 patients with classic galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analzyed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test. PMID:20863731

  8. Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies.

    PubMed

    Li, Yijun; Ptolemy, Adam S; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T

    2011-01-01

    The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype-phenotype relationships, we had developed an assay for GALT activity alone using LC-MS/MS. In this study we generated a robust and sensitive LC-MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([(13)C(6)]-uridine diphosphate galactose in GALT assay and [(13)C(6)]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC-MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4±4.2 and GALK activity of 1.8±0.47 (mean±SD) μmol⋅(g Hgb)(-1) h(-1). Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analyzed. Lastly, we tested the feasibility of adapting this LC-MS/MS based GALT/GALK assay as a newborn screening (NBS) test. PMID:20863731

  9. Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae.

    PubMed

    Meyer, J; Walker-Jonah, A; Hollenberg, C P

    1991-11-01

    We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae. PMID:1922058

  10. Regional brain distribution of 2-deoxy-2-[F-18]Fluoro-D-talose: A new PET tracer for measurement of galactokinase activity

    SciTech Connect

    Haradahira, T.; Inoue, O.; Suzuki, K.

    1994-05-01

    We have recently developed a 2-deoxy-2-[F-18]fluoro-D-talose (2-[F-18]FDTal) as a new PET tracer for measurements of galactokinase activities in tissues. The rational of 2-[F-18]FDTal as a PET tracer is based on the metabolic trapping by a formation of 2-[F-18]FDTal-1-phosphate by galactokinase. In this study, we have examined the regional brain distribution of 2-[F-18]FDTal in monkey by PET, and compared it with those of 2-deoxy-2-[F-18]fluoro-D-glucose (2-[F-18]FDG) and 2-deoxy-2-[F-18]fluoro-D-galactose (2-[F-18]FDTal), PET tracers for D-glucose and D-galactose metabolisms, respectively. The F-18 sugars used for the PET studies were prepared through nucleophilic fluorinations of the corresponding triflates with [F-18]fluoride. PET imaging obtained by i.v. injection of 2-[F-18]FDTal in rhesus monkey showed very high accumulation of the radioactivity into an occipital cortex region. This regional distribution was very similar to that of 2-[F-18]FDGal, but was quite different with that of 2-[F-18]FDG. In PET data analyses, washout of the radioactivity from the occipital cortex (30% loss of the initial activity) was observed in an early period ({<=}5 min) after injection of 2-[F-18]FDTal, in contrast with the continuous increase of the radioactivity in the same region after injection of 2-[F-18]FDGal. This data indicates a smaller phosphorylation rate constant (K3) of 2-[F-18]FDTal by brain galactokinase than that of 2-[F-18]FDGal. 2-[F-18]FDGal has been reported to be partly metabolized into an UDP-2-[F-18]FDGal via 2-CF-18]FDGal-l-phosphate in animal brains. Therefore 2-[F-18]FDTal offers an advantage over 2-[F-18]FDGal in undergoing its simple metabolism which enables us to make a simple kinetic model for PET imaging. We conclude that 2-[F-18]FDTal may be a new PET tracer to give a characteristic regional brain distribution which reflect the regional galactokinase activity.

  11. The gene of an early onset progressive cataract (cerulean cataract) maps to 17q24

    SciTech Connect

    Armitage, M.M.; Ferrell, R.E.; Kivlin, J.D.

    1994-09-01

    Cerulean cataract is an autosomal dominant, fully penetrant, early onset, progressive cataract characterized by blue or white opacifications in the nucleus and cortex of the lens. A five generation family with 44 available affected members in three generations allowed exclusion of linkage of the cerulean cataract phenotype to lens structural protein genes and to all of the chromosomal regions to which autosomal dominant cataract phenotypes have previously been mapped. Exclusion of the plausible candidate instigated a genome-wide search utilizing short tandem repeat polymorphims. The genome search localized the cerulean cataract disease gene to chromosomal region 17q24. The three markers closest to the disease gene are D17S802 [Z({theta})=9.20 at ({theta})=0.086], D17S836 [Z({theta})=4.22 at ({theta})=0.061], and AFMa238yb5 [Z({theta})=7.11 at ({theta})=0.032]. Multipoint analysis yielded a maximum lod score of Z({theta})=11.4 between D17S802 and D17S836 at recombination rates of 0.048 and 0.013 respectively. Three genes that map near the 17q24 chromosomal region and are known to contain highly polymorphic microsatellites were tested for linkage. The genes, DHP-sensitive calcium channel gamma subunit (CACNLG), human somatastatin receptor (SSTR2), and the skeletal muscle sodium channel alpha subunit (SCN4A), were all excluded [Z({theta})=-{infinity} at ({theta})=0] as the gene causing cerulean cataract. The galactokinase (GK1) gene has not been cloned, but its map location is 17q23-q25. Galactokinase deficiency is characterized by a recessive, progressive, early onset cataract. Because of the map location of galactokinase, the age-at-onset, and progressive nature of cataracts associated with galactokinase deficiency, galactokinase is being investigated as a candidate gene for the cerulean cataract phenotype.

  12. Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures

    PubMed Central

    Thomas, Terence D.; Crow, Vaughan L.

    1984-01-01

    Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal−). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal− cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal+ organisms eventually took over the culture with all four strains examined. Gal+ cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal+ organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-β-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal+-Gal− cultures were repeatedly transferred in milk, the Gal− cells became the dominant cell type. The Gal− phenotype of stock cultures probably reflects their prolonged maintenance in milk. PMID:16346586

  13. Isolation of mutations that act in trans to alter expression from a yeast hsp70 promoter.

    PubMed Central

    Findly, R C; Alavi, H; Platt, T

    1988-01-01

    Transcription of SSA1 (formerly YG100), a member of the hsp70 gene family in Saccharomyces cerevisiae, increases dramatically upon heat shock. An expression vector in which the promoter of SSA1 is fused to the Escherichia coli galactokinase gene (galK) was constructed and transformed into a galactokinase-deficient yeast strain. The transformants grew on galactose at 23 degrees C, but increased expression of the SSA1-galK fusion gene inhibited growth of cells on galactose at 37 degrees C. Selection for survivors under nonpermissive conditions yielded a class of mutants, termed HSR (for heat shock regulation), which showed reduced levels of expression of the hsp70-galK gene fusion as determined by measurement of galactokinase activity. Similar effects on beta-galactosidase activity were obtained when an SSA1-lacZ fusion vector was introduced into the mutants, suggesting action in trans through the SSA1 promoter. Analysis of Northern (RNA) blots demonstrated that the reduction in expression was a result of decreased mRNA levels for the fusion gene. In addition, mRNA levels of the endogenous SSA1 gene are reduced in an HSR mutant. Genetic analysis has shown that these mutations act in trans and affect both transcription from the SSA1 promoter and turnover of the fusion transcript. These are the first trans-acting mutations known to affect directly the transcriptional regulation and transcript stability of heat shock genes in eucaryotes. Images PMID:3145411

  14. Saccharomyces cerevisiae mutants resistant to catabolite repression: use in cheese whey hydrolysate fermentation

    SciTech Connect

    Bailey, R.B.; Benitez, T.; Woodward, A.

    1982-09-01

    Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant. (Refs. 23).

  15. Sequence of the Saccharomyces GAL region and its transcription in vivo.

    PubMed Central

    Citron, B A; Donelson, J E

    1984-01-01

    In Saccharomyces, the enzymes used to convert galactose to glucose are specified by three coordinately expressed, tightly linked genes, GAL7, GAL10, and GAL1. These genes are induced by galactose and are controlled by the positive regulator gene gal4 and the negative regulator gene gal80. GAL81 mutations, which are known to alter the gal4 protein, produce a constitutive phenotype. We have cloned fragments of Saccharomyces carlsbergensis DNA that span 26.3 kilobases surrounding the three clustered GAL genes. About 5 kilobases of the sequence was determined, which includes the entire GAL1 gene, the two intercistronic regions, and portions of the coding sequences of GAL10 and GAL7. Some amino acid homology between the GAL1 gene product, galactokinase, and the Escherichia coli galactokinase was detected. By using various Saccharomyces DNA fragments, the accumulation of GAL1 and GAL10 RNA in yeast cells after induction with galactose was studied. Our results, using wild-type, gal4-, gal80-, and GAL81-1- yeast cells, support the hypothesis that control is exerted at the transcriptional level. Images PMID:6715281

  16. Improved one-pot multienzyme (OPME) systems for synthesizing UDP-uronic acids and glucuronides†

    PubMed Central

    Muthana, Musleh M.; Qu, Jingyao; Xue, Mengyang; Klyuchnik, Timofey; Siu, Alex; Li, Yanhong; Zhang, Lei; Yu, Hai; Li, Lei; Wang, Peng G.

    2015-01-01

    Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and shown to be able to use various uronic acids as substrates to produce the corresponding uronic acid-1-phosphates. AtGlcAK or Bifidobacterium infantis galactokinase (BiGalK) was used with a UDP-sugar pyrophosphorylase, an inorganic pyrophosphatase, with or without a glycosyltransferase for highly efficient synthesis of UDP-uronic acids and glucuronides. These improved cost-effective one-pot multienzyme (OPME) systems avoid the use of nicotinamide adenine dinucleotide (NAD+)-cofactor in dehydrogenase-dependent UDP-glucuronic acid production processes and can be broadly applied for synthesizing various glucuronic acid-containing molecules. PMID:25686901

  17. Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.

    PubMed Central

    Zitomer, R S; Walthall, D A; Rymond, B C; Hollenberg, C P

    1984-01-01

    A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results. PMID:6390186

  18. A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species 1

    PubMed Central

    Gross, Kenneth C.; Pharr, David M.

    1982-01-01

    Conversion of [14C]galactose (Gal) 1-P, UDP-[14C]Gal, or UDP-[14C]glucose to [14C]sucrose was observed when cell-free homogenates of cucumber (Cucumis sativus L.) fruit peduncles were incubated with individual 14C-labeled substrates, appropriate cofactors, and fructose. The sucrose product was labeled only in the glucose moiety. Conversion of [14C]Gal-1-P to [14C]sucrose was catalyzed by extracts of peduncles from all other stachyose transporting species tested, as well as green bean (a sucrose transporter) but was not catalyzed by peduncle extracts from three other sucrose transporting species. In cucumber, the ability of extracts to form [14C]sucrose from [14C]Gal-1-P was greater when peduncles were harvested from growing fruit than from unpollinated ovaries. [14C]Sucrose formation from [14C]Gal-1-P was inhibited by Mg · PPi, Mg · UDP, UMP, and sucrose. α-Galactosidase, galactokinase, UDP-gal pyrophosphorylase, UDP-Gal-4′-epimerase, UDP-glucose pyrophosphorylase, and sucrose synthase activities were detected in peduncle extracts. Neither sucrose phosphate synthetase nor hexose-1-P uridyltransferase were detected. Peduncle tissue contained a small pool of free galactose. These results suggest a potential pathway for the metabolism of galactose moieties hydrolyzed from stachyose, the major sugar transported by cucumber plants. PMID:16662141

  19. Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey.

    PubMed

    Barrett, Eoin; Stanton, Catherine; Zelder, Oskar; Fitzgerald, Gerald; Ross, R Paul

    2004-05-01

    The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and beta-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited beta-galactosidase activity in excess of 1000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and beta-galactosidase genes exhibited beta-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability. PMID:15128544

  20. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

    SciTech Connect

    Yaung, Stephanie J.; Deng, Luxue; Li, Ning; Braff, Jonathan L.; Church, George M.; Bry, Lynn; Wang, Harris H.; Gerber, Georg K.

    2015-03-11

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Here, our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

  1. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

    DOE PAGESBeta

    Yaung, Stephanie J.; Deng, Luxue; Li, Ning; Braff, Jonathan L.; Church, George M.; Bry, Lynn; Wang, Harris H.; Gerber, Georg K.

    2015-03-11

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Populationmore » dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Here, our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.« less

  2. Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

    PubMed Central

    Yaung, Stephanie J; Deng, Luxue; Li, Ning; Braff, Jonathan L; Church, George M; Bry, Lynn; Wang, Harris H; Gerber, Georg K

    2015-01-01

    Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo. PMID:25762151

  3. Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

    PubMed Central

    Peterson, J L; McBride, O W

    1980-01-01

    The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity. Images PMID:6929511

  4. The gal Genes for the Leloir Pathway of Lactobacillus casei 64H

    PubMed Central

    Bettenbrock, Katja; Alpert, Carl-Alfred

    1998-01-01

    The gal genes from the chromosome of Lactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of the L. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking the HindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKp promoter is inducible by galactose in the medium, while galEp constitutes a semiconstitutive promoter located in galK. PMID:9603808

  5. Glutamine synthetase-constitutive mutation affecting the glnALG upstream promoter of Escherichia coli.

    PubMed Central

    León, P; Romero, D; Garciarrubio, A; Bastarrachea, F; Covarrubias, A A

    1985-01-01

    The spontaneous gln-76 mutation of Escherichia coli (Osorio et al., Mol. Gen. Genet. 194:114-123, 1984) was previously shown to be responsible for the cis-dominant constitutive expression of the glnA gene in the absence of a glnG-glnF activator system. Nucleotide sequence analysis has now revealed that gln-76 is a single transversion T.A to A.T, an up-promoter mutation affecting the -10 region of glnAp1, the upstream promoter of the glnALG control region. Both, wild-type and gln-76 DNA control regions were cloned into the promoter-probe plasmid pKO1. Galactokinase activity determinations of cells carrying the fused plasmids showed 10-fold more effective expression mediated by gln-76 than by the glnA wild-type control region. Primer extension experiments with RNA from strains carrying the gln-76 control region indicated that the transcription initiation sites were the same in both the gln-76 mutant and the wild type. Images PMID:2866175

  6. Regulation of Salmonella typhimurium ilvYC genes.

    PubMed

    Blazey, D L; Burns, R O

    1984-09-01

    The Salmonella typhimurium LT2 ilvYC genes were studied by fusion of each gene to the Escherichia coli K-12 galK gene. The expression of ilvY and ilvC could then be determined by measurement of the galK-encoded galactokinase enzyme. The promoter for ilvC, pC, was located by this technique to a 0.42-kilobase BglII-EcoRI fragment of the S. typhimurium ilvGEDAYC gene cluster. This sequence was completely sufficient for alpha-acetohydroxyacid-inducible galK expression. The ilvY gene was located within a 1.0-kilobase XhoI-SalI fragment. ilvY gene expression was constitutive with respect to ilv-specific control signals. The ilvY gene was transcribed in the same direction as the other two transcriptional units in the ilvGEDAYC gene cluster, ilvGEDA and ilvC. Transcription of the ilvC gene was completely dependent upon the activity of its own promoter, pC, and independent from transcription of the ilvY gene. The role of the intervening region between ilvY and ilvC in regulation of ilvC expression was explored. PMID:6090400

  7. Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol.

    PubMed

    Majumder, Avishek; Sultan, Abida; Jersie-Christensen, Rosa R; Ejby, Morten; Schmidt, Bjarne Gregers; Lahtinen, Sampo J; Jacobsen, Susanne; Svensson, Birte

    2011-09-01

    Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included β-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4-epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol. PMID:21751373

  8. Innovative Therapy for Classic Galactosemia - Tale of Two HTS

    PubMed Central

    Tang, M; Odejinmi, SI; Vankayalapati, H; Wierenga, K; Lai, K

    2011-01-01

    Classic Galactosemia is an autosomal recessive disorder caused by the deficiency of galactose-1-phosphate uridylyltransferase (GALT), one of the key enzymes in the Leloir pathway of galactose metabolism. While the neonatal morbidity and mortality of the disease are now mostly prevented by newborn screening and galactose restriction, long-term outcome for older children and adults with this disorder remains unsatisfactory. The pathophysiology of Classic Galactosemia is complex, but there is convincing evidence that galactose-1-phosphate (gal-1P) accumulation is a major, if not the sole pathogenic factor. Galactokinase (GALK) inhibition will eliminate the accumulation of gal-1P from both dietary sources and endogenous production, and efforts towards identification of therapeutic small molecule GALK inhibitors are reviewed in detail. Experimental and computational high-throughput screenings of compound libraries to identify GALK inhibitors have been conducted, and subsequent studies aimed to characterize, prioritize, as well as to optimize the identified positives have been implemented to improve the potency of promising compounds. Although none of the identified GALK inhibitors inhibit glucokinase and hexokinase, some of them cross-inhibit other related enzymes in the GHMP small molecule kinase superfamily. While this finding may render the on-going hit-to-lead process more challenging, there is growing evidence that such cross-inhibition could also lead to advances in antimicrobial and anti-cancer therapies. PMID:22018723

  9. Galactose metabolism by Streptococcus mutans.

    PubMed

    Abranches, Jacqueline; Chen, Yi-Ywan M; Burne, Robert A

    2004-10-01

    The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons. PMID:15466549

  10. Galactose and Lactose Genes from the Galactose-Positive Bacterium Streptococcus salivarius and the Phylogenetically Related Galactose-Negative Bacterium Streptococcus thermophilus: Organization, Sequence, Transcription, and Activity of the gal Gene Products

    PubMed Central

    Vaillancourt, Katy; Moineau, Sylvain; Frenette, Michel; Lessard, Christian; Vadeboncoeur, Christian

    2002-01-01

    Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (β-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3′ end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities. PMID:11790749

  11. Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model

    PubMed Central

    Jumbo-Lucioni, Patricia; Parkinson, William; Broadie, Kendal

    2014-01-01

    Classic galactosemia (CG) is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT), which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP)-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG), showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT) greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ). Dietary galactose and mutation of galactokinase (dGALK) or UDP-glucose dehydrogenase (sugarless) genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP) moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG) co-receptor and Wnt ligand

  12. The molecular basis of galactosemia - Past, present and future.

    PubMed

    Timson, David J

    2016-09-10

    Galactosemia, an inborn error of galactose metabolism, was first described in the 1900s by von Ruess. The subsequent 100years has seen considerable progress in understanding the underlying genetics and biochemistry of this condition. Initial studies concentrated on increasing the understanding of the clinical manifestations of the disease. However, Leloir's discovery of the pathway of galactose catabolism in the 1940s and 1950s enabled other scientists, notably Kalckar, to link the disease to a specific enzymatic step in the pathway. Kalckar's work established that defects in galactose 1-phosphate uridylyltransferase (GALT) were responsible for the majority of cases of galactosemia. However, over the next three decades it became clear that there were two other forms of galactosemia: type II resulting from deficiencies in galactokinase (GALK1) and type III where the affected enzyme is UDP-galactose 4'-epimerase (GALE). From the 1970s, molecular biology approaches were applied to galactosemia. The chromosomal locations and DNA sequences of the three genes were determined. These studies enabled modern biochemical studies. Structures of the proteins have been determined and biochemical studies have shown that enzymatic impairment often results from misfolding and consequent protein instability. Cellular and model organism studies have demonstrated that reduced GALT or GALE activity results in increased oxidative stress. Thus, after a century of progress, it is possible to conceive of improved therapies including drugs to manipulate the pathway to reduce potentially toxic intermediates, antioxidants to reduce the oxidative stress of cells or use of "pharmacological chaperones" to stabilise the affected proteins. PMID:26143117

  13. Molecular and Biochemical Analysis of the Galactose Phenotype of Dairy Streptococcus thermophilus Strains Reveals Four Different Fermentation Profiles

    PubMed Central

    de Vin, Filip; Rådström, Peter; Herman, Lieve; De Vuyst, Luc

    2005-01-01

    Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related. PMID:16000774

  14. A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability*

    PubMed Central

    Guo, Qin; Zhang, Wei; Ma, Liu-liu; Chen, Qi-he; Chen, Ji-cheng; Zhang, Hong-bo; Ruan, Hui; He, Guo-qing

    2010-01-01

    The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer. PMID:20043351

  15. Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model.

    PubMed

    Jumbo-Lucioni, Patricia; Parkinson, William; Broadie, Kendal

    2014-12-01

    Classic galactosemia (CG) is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT), which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP)-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG), showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT) greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ). Dietary galactose and mutation of galactokinase (dGALK) or UDP-glucose dehydrogenase (sugarless) genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP) moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG) co-receptor and Wnt ligand levels

  16. Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of Streptococcus salivarius

    PubMed Central

    Plamondon, Pascale; Brochu, Denis; Thomas, Suzanne; Fradette, Julie; Gauthier, Lucie; Vaillancourt, Katy; Buckley, Nicole; Frenette, Michel; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His15) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser46) by an ATP-dependent protein kinase (His∼P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His15 by EI nor the phosphorylation at Ser46 by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His∼P) than the wild-type strain, while the levels of free HPr and HPr(His∼P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of α-galactosidase, β-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule. PMID:10559156

  17. Diversity of Streptococcus salivarius ptsH Mutants That Can Be Isolated in the Presence of 2-Deoxyglucose and Galactose and Characterization of Two Mutants Synthesizing Reduced Levels of HPr, a Phosphocarrier of the Phosphoenolpyruvate:Sugar Phosphotransferase System

    PubMed Central

    Thomas, Suzanne; Brochu, Denis; Vadeboncoeur, Christian

    2001-01-01

    In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for α-galactosidase, β-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of α-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and

  18. Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.

    PubMed Central

    Marczak, J E; Brandriss, M C

    1989-01-01

    The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting