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PubMed2000

Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

Aldose-Ketose Isomerases↗

PubMed2001

Improvement of tagatose conversion rate by genetic evolution of thermostable galactose isomerase.

To enhance the isomerization rate of galactose into tagatose, a thermostable galactose isomerase, which was isolated from bacteria growing in a hot spring, was genetically improved using the error-prone PCR method. From 500 mutant clones, a clone showing improved conversion activity was selected. The sequence of the selected clone had five amino acid changes: His(228)-->Asp, Gly(384)-->Asp, Ser(393)-->Thr, Lys(428)-->Asn and Asp(475)-->Lys. The improved galactose isomerase had an 11-fold higher reaction rate than the original.

Aldose-Ketose Isomerases↗

PubMed1970

Frog brain uridine diphosphate galactose-N-acetylgalactosaminyl-N-acetylneuraminylgalactosylglucosylceramide galactosyltransferase.

1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1-->4)-N-acetylneuraminyl-(2-->3)-galactosyl-(1-->4)-glucosylceramide (G(M2)) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose-G(M2) galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11(1/2)) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2-7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn(2+) for maximal activity and the K(m) for Mn(2+) was determined as 2.2mm. The half-maximal velocity was obtained at a G(M2) concentration of 0.18mm. Inhibition of the enzymic reaction was found when the G(M2) concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed.

Animals↗

PubMed1971

Galactose-specific messenger ribonucleic acid contents in Escherichia coli.

A method is described for measuring the proportion of galactose-specific mRNA (gal-mRNA) in the total RNA extracted from pulse-labelled cells of Escherichia coli K12, by DNA-RNA hybridization with DNA prepared from bacteriophage lambdadg. RNA from wild-type E. coli was compared with RNA from a homogenote carrying the gal operon both in the chromosome and in a substituted sex-factor, and with RNA from a deletion strain that carried the galactose operon only in the exogenote. In each case the cultures were induced with fucose. Under these conditions the amount of gal-mRNA was found to be proportional to the content of galactokinase in the different cultures, and to the gene frequency. The amounts of gal-mRNA in an O(c) mutant and an R(-) mutant were also proportional to the observed contents of galactokinase. In cultures repressed for the enzymes of the galactose operon with thiomethylgalactoside, the content of gal-mRNA was higher than expected from the content of galactokinase. Possible explanations of this finding are discussed.

Chromosomes, Bacterial↗

PubMed1974

Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme.

The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein-N-acetylglucosamine and alpha-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn(2+) ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site.

Centrifugation, Density Gradient↗

PubMed2006

Participation of a galactose-specific C-type lectin in Drosophila immunity.

A galactose-specific C-type lectin has been purified from a pupal extract of Drosophila melanogaster. This lectin gene, named DL1 (Drosophila lectin 1), is part of a gene cluster with the other two galactose-specific C-type lectin genes, named DL2 (Drosophila lectin 2) and DL3 (Drosophila lectin 3). These three genes are expressed differentially in fruit fly, but show similar haemagglutinating activities. The present study characterized the biochemical and biological properties of the DL1 protein. The recombinant DL1 protein bound to Escherichia coli and Erwinia chrysanthemi, but not to other Gram-negative or any other kinds of microbial strains that have been investigated. In addition, DL1 agglutinated E. coli and markedly intensified the association of a Drosophila haemocytes-derived cell line with E. coli. For in vivo genetic analysis of the lectin genes, we also established a null-mutant Drosophila. The induction of inducible antibacterial peptide genes was not impaired in the DL1 mutant, suggesting that the galactose-specific C-type lectin does not participate in the induction of antibacterial peptides, but possibly participates in the immune response via the haemocyte-mediated mechanism.

Amino Acid Sequence↗

PubMed1995

Kinetics and thermodynamics of the binding of forskolin to the galactose-H+ transport protein, GalP, of Escherichia coli.

The binding of the transport inhibitor, forskolin, to the galactose-H+ symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 microM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.9-1.4 microM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (kon) and dissociation (koff) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 microM-1.s-1 and 5.1-11.5 s-1 respectively, and the Kd was calculated to be 1.5 microM. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by sugar provided an additional method to calculate the dissociation rate constant for forskolin (koff = 12.4-13.0 s-1). The rate of the slow change in protein fluorescence (3-5 s-1) was independent of the forskolin concentration, indicating an isomerization of the transporter between different conformations, possibly outward- and inward-facing forms. These kinetic parameters were determined at a series of temperatures, so that the thermodynamics of forskolin binding and transporter re-orientation could be analysed. The binding process was entropically driven (delta S = 83.7 J.K-1.mol-1; delta H = 8.25 kJ.mol-1), similar to that for cytochalasin B, which is also an inhibitor of GalP. Measurements of the binding of [3H]forskolin by equilibrium dialysis revealed competitive displacement of bound forskolin by cytochalasin B, possibly suggesting that the sugar, forskolin and cytochalasin B binding sites are overlapping; the Kds for forskolin and cytochalasin B were calculated to be 0.85 microM and 4.77 microM respectively, and the concentration of binding sites was 10.2 nmol.mg-1.

Bacterial Proteins↗

PubMed1996

A galactose-rich, cell-wall glycoprotein from styles of Nicotiana alata.

A basic, galactose-rich style glycoprotein (GaRSGP) encoded by a previously characterized style-specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45-120 kDa on SDS-PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline-rich glycoproteins (P/HRGPs) to be characterized both as a cDNA-clone and protein. Glycans are attached to the protein backbone through both O- and N-glycosidic linkages with the majority of the carbohydrate being O-linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style-specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style-specific carbohydrate epitope(s) which may play a role in style function.

Amino Acid Sequence↗

PubMed2002

Galactosyl-mimodye ligands for Pseudomonas fluorescens beta-galactose dehydrogenase.

Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-biomimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appropriate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic 'linker' molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature 'spacer' molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Following their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensbeta-galactose dehydrogenase. In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye-ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.

Amino Acid Sequence↗

PubMed2002

Molecular characterization of MRG19 of Saccharomyces cerevisiae. Implication in the regulation of galactose and nonfermentable carbon source utilization.

We have reported previously that multiple copies of MRG19 suppress GAL genes in a wild-type but not in a gal80 strain of Saccharomyces cerevisiae. In this report we show that disruption of MRG19 leads to a decrease in GAL induction when S. cerevisiae is induced with 0.02% but not with 2.0% galactose. Disruption of MRG19 in a gal3 background (this strain shows long-term adaptation phenotype) further delays the GAL induction, supporting the notion that its function is important only under low inducing signals. As a corollary, disruption of MRG19 in a gal80 strain did not decrease the constitutive expression of GAL genes. These results suggest that MRG19 has a role in GAL regulation only when the induction signal is weak. Unlike the effect on GAL gene expression, disruption of MRG19 leads to de-repression of CYC1-driven beta-galactosidase activity. MRG19 disruptant also showed a twofold increase in the rate of oxygen uptake as compared with the wild-type strain. ADH2, CTA1, DLD1, and CYC7 promoters that are active during nonfermentative growth did not show any de-repression of beta-galactosidase activity in the MRG19 disruptant. Western blot analysis indicated that MRG19 is a glucose repressible gene and is expressed in galactose and glycerol plus lactate. Experiments using green fluorescent protein fusion constructs indicate that Mrg19p is localized in the nucleus consistent with the presence of a consensus nuclear localization signal sequence. Based on the above results, we propose that Mrg19p is a regulator of galactose and nonfermentable carbon utilization.

Base Sequence↗

PubMed2003

Crystal structure at 3 A of mistletoe lectin I, a dimeric type-II ribosome-inactivating protein, complexed with galactose.

The X-ray structure of mistletoe lectin I (MLI), a type-II ribosome-inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 A resolution to an R-factor of 19.9% using 21 899 reflections, with Rfree 24.0%. MLI forms a homodimer (A-B)2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N-terminal domains of two B-chains involving weak polar and non-polar interactions. Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 A apart on one side of the dimer, and two C-terminal sugar-binding sites are 87 A apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3-OH and 4-OH and hydrophobic contact by an aromatic ring. In addition, at the N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydrophobic contact. MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from a two-domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.

Adjuvants, Immunologic↗

PubMed1971

Uridine diphosphate-4-keto-glucose, an intermediate in the uridine diphosphate-galactose-4-epimerase reaction.

When UDP-galactose 4-epimerase (EC 5.1.3.2) from Escherichia coli is incubated with UDP-galactose, then reduced with NaB(3)H(4), label is found in UDP-glucose and UDP-galactose. Enzymatic and chemical degradation demonstrates that the label is bound to carbon 4 of the glycosyl moieties. These results provide direct evidence for the existence of UDP-4-keto-glucose as an enzyme-bound intermediate in the epimerization reaction, and they exclude the formation of a 3-keto intermediate.

Alcohol Oxidoreductases↗

PubMed1972

Enhancement of hexose entry into chick fibroblasts by starvation: differential effect on galactose and glucose.

Glucose entry, as measured by 5-min uptake into the acid-soluble fraction, is enhanced 15-30 times by long-term (12-24 hr) hexose starvation of chick fibroblasts. The rate of galactose accumulation in the cells increases only 5 times under the same conditions of starvation. Several carbon and energy sources that were tested for their effect on this "derepression" can be classified as: (i) those resembling glucose in blocking the "stimulation," (ii) those permitting full "derepression"; and (iii) those partially preventing the enhanced entry. Inhibitors of protein synthesis block enhancement under conditions otherwise conducive to it. We conclude that the glucose and galactose carrier systems are not identical, based largely on the asymmetric "repression" observed when glucose and galactose are compared as "repressors."

Animals↗

PubMed1987

Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon.

Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, galP1 and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galP1 is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the galT, galE, and galK genes. The second promoter, galP2, is located within the operon just upstream of the galE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.

Base Sequence↗

PubMed1996

A disulfide-bonded dimer of the Golgi beta-galactoside alpha2,6-sialyltransferase is catalytically inactive yet still retains the ability to bind galactose.

The alpha2,6-sialyltransferase is a terminal glycosyltransferase localized in the trans Golgi and trans Golgi network. Here we show that 30% of the total rat liver Golgi alpha2,6-sialyltransferase forms a disulfide-bonded 100-kDa species that can be converted to the 50-kDa monomer form of the enzyme upon reduction. Limited proteolysis of both enzyme forms demonstrates that the 100-kDa species is a disulfide-bonded homodimer of the alpha2,6-sialyltransferase. The alpha2,6-sialyltransferase disulfide-bonded dimer is found in bovine liver Golgi membranes and in Golgi membranes prepared and solubilized in the presence of 100 mM iodoacetamide, suggesting that it is not unique to rat liver or formed aberrantly upon membrane lysis. The dimer form of the enzyme possesses no significant catalytic activity and has a much lower affinity for CDP-hexanolamine-agarose compared with the monomer form. In contrast, both the alpha2,6-sialyltransferase monomer and the disulfide-bonded dimer bind strongly to galactose and galactose-terminated substrates. These results suggest that the alpha2,6-sialyltransferase disulfide-bonded dimer lacks catalytic activity due to a weak affinity for its sugar nucleotide donor, CMP-NeuAc, and that this catalytically inactive form of the enzyme may act as a galactose-specific lectin in the Golgi.

Animals↗

PubMed1999

Using a galactose library for exploration of a novel hydrophobic pocket in the receptor binding site of the Escherichia coli heat-labile enterotoxin.

The binding of the B subunits of Escherichia coli heat-labile enterotoxin (LT) to epithelial cells lining the intestines is a critical step for the toxin to invade the host. This mechanism suggests that molecules which possess high affinity to the receptor binding site of the toxin would be good leads for the development of therapeutics against LT. The natural receptor for LT is the complex ganglioside GM1, which has galactose as its terminal sugar. A chemical library targeting a novel hydrophobic pocket in the receptor binding site of LT was constructed based on galactose derivatives and screened for high affinity to the receptor binding site of LT. This screening identified compounds that have 2-3 orders of magnitude higher affinity toward the receptor binding site of LT than the parent compound, galactose. The present findings will pave the way for developing simple and easily synthesizable molecules, instead of complex oligosaccharides, as drugs and/or prophylactics against LT-caused disease.

Bacterial Toxins↗

PubMed2001

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.

Binding Sites↗

PubMed2003

Crystal structure of rice alpha-galactosidase complexed with D-galactose.

alpha-Galactosidases catalyze the hydrolysis of alpha-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice alpha-galactosidase has been determined at 1.5A resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of alpha-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (beta/alpha)8-barrel structure, and the C-terminal domain was made up of eight beta-strands containing a Greek key motif. The structure was solved as a complex with d-galactose, providing a mode of substrate binding in detail. The d-galactose molecule was found bound in the active site pocket on the C-terminal side of the central beta-barrel of the catalytic domain. The d-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice alpha-galactosidase with chicken alpha-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes.

Amino Acid Sequence↗