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PubMed1996

The galE gene encoding the UDP-galactose 4-epimerase of Brevibacterium lactofermentum is coupled transcriptionally to the dmdR gene.

The galE gene of Brevibacterium lactofermentum, encoding UDP-galactose 4-epimerase (EC 5.1.3.2), has been identified by DNA sequencing downstream from the orf1-sigB-dmdR region. The arrangement of the sigB-dtxR-galE cluster is also conserved in Corynebacterium diphtheriae. The deduced galE product was a protein of 329 aa residues (35.4 kDa) that shared a high degree of identity to known UDP-galactose 4-epimerase proteins from Gram-positive microorganisms (Streptomyces lividans and Streptococcus thermophilus). Transcriptional analysis of the dmdR and galE genes in nutrient-rich medium showed that these genes are part of an operon, that is actively transcribed as a bicistronic mRNA during the exponential growth phase, but transcription of the operon is decreased during the stationary growth phase. In addition, the dmdR gene was also expressed as a monocistronic 0.7-kb transcript during the active growth phase.

Bacterial Proteins↗
PubMed2006

An efficient synthesis of quinoxaline derivatives from 4-chloro-4-deoxy-alpha-D-galactose and their cytotoxic activities.

A novel and efficient method for the synthesis of quinoxaline derivatives has been developed. Isopropylidenation of 4-chloro-4-deoxy-alpha-D-galactose with 2,2-dimethoxypropane, followed by selective hydrolysis, afforded 2,3-O-isopropylidene-4-chloro-4-deoxy-D-galactose di-methyl acetal (3) as a sole product. Oxidation of compound 3 with (Bu3Sn)2O-Br2 gave corresponding hex-5-ulose derivative in high yields. The hex-5-ulose derivative reacted with o-phenylenediamines under neutral conditions to afford quinoxaline derivatives in reasonable yields. The in vitro cytotoxic activities of these quinoxaline derivatives were investigated.

Antineoplastic Agents↗
PubMed2004

UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site.

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.

Catalysis↗
PubMed2005

Purification and characterization of a soluble form of the recombinant human galactose-beta1,3-glucuronosyltransferase I expressed in the yeast Pichia pastoris.

The galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) catalyzes the transfer of glucuronic acid from UDP-alpha-D-glucuronic acid onto the terminal galactose of the trisaccharide glycosaminoglycan-protein linker region of proteoglycans. This enzyme plays a key role in the process of proteoglycan assembly since the completion of the linkage region is essential for the conversion of a core protein into a functional proteoglycan. To investigate the enzymatic properties of human GlcAT-I, we established an expression system for producing a soluble form of enzyme in the methylotrophic yeast Pichia pastoris and developed a three-step purification procedure using a combination of anion exchange, cation exchange and heparin chromatographies. This procedure yielded 1.6 mg homogeneous enzyme from 200 ml yeast cell culture, with a specific activity value of 1.5 micromol/min/mg protein. Analysis of the specificity of GlcAT-I towards Galbeta1-3Gal and Galbeta1-4GlcNAc derivatives known as substrates of the beta1,3-glucuronosyltransferases, showed that the enzyme exhibited a strict selectivity towards Galbeta1-3Gal structures. Thus, the large source of purified active enzyme allowed the determination of the kinetic parameters of GlcAT-I towards the donor substrate UDP-GlcA and the acceptor substrate digalactoside Galbeta1-3Gal.

Cloning, Molecular↗
PubMed2002

Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins.

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.

Amino Acid Sequence↗
PubMed1998

Newborn screening for galactosemia: ultramicro assay for galactose-1-phosphate-uridyltransferase activity.

An enzymatically optimized, miniaturized (20 microl) fluorimetric assay of galactose-1-phosphate-uridyltransferase using dried blood spots for newborn screening is presented. The Beutler reaction principle has been adapted to the microtiter plate technology and acetone/methanol was used for complete deproteinization. A special ultramicro multiwell screening plate resistant to organic solvents has been developed and employed. The assay is simple, sensitive and inexpensive, due to small reagent volumes and the low prices of ultramicro screening plates. The reaction is linear with galactose-1-phosphate-uridyltransferase activity up to 120 min of incubation time. It shows low imprecision and good correlation to a quantitative validation test. For standardization the use of plate means or medians of activity or fluorescence values is proposed. Individual blank measurement prevents false negative assessments.

Fluorometry↗
PubMed1998

Differential UDP-galactose-4'-epimerase (GALE) enzymatic activity and mRNA expression in the rat mammary gland during lactation.

We have investigated the UDP-galactose-4'-epimerase (GALE) enzymatic activity and mRNA expression in the rat mammary gland during lactation. We report a dramatic increase in the GALE enzymatic activity correlated with an increase in the mRNA transcript expression. These results indicate a transcriptional regulation of the enzyme during lactation in the rat mammary gland. Our data are of double interest for further investigation: first, the mammary gland provides a suitable model for the characterisation of the transcriptional regulation elements of GALE which are still unknown in mammals; second, GALE expression could help to compensate UDP-galactose deficiency in classic galactosaemia.

Animals↗
PubMed1995

Myelodysplasia and deficiency of uridine diphosphate-galactose 4-epimerase.

A 4-year-old girl known to have peripheral uridine diphosphate-galactose 4-epimerase deficiency was examined for bruising and thrombocytopenia. She had dysplastic peripheral blood and bone marrow changes, with a global platelet function defect. Uridine diphosphate-galactose-4-epimerase participates in a metabolic pathway that provides substrates for posttranslational glycosylation of secreted and membrane glycoproteins, including hematopoietic growth factors and their receptors; there may be a causal relationship between the two disorders.

Bone Marrow↗
PubMed1997

Black children deficient in galactose 1-phosphate uridyltransferase: correlation of activity and immunoreactive protein in erythrocytes and leukocytes.

A recent study found a high prevalence of a missense mutation (S135L) in the gene for galactose 1-phosphate uridyltransferase (GALT) in black children with galactosemia (J Pediatr 1996; 128:89-95). In the present study, GALT activity and GALT protein content were measured in erythrocytes and leukocytes of eight black and seven white galactosemic (GALT-deficient) children, for correlation with the presence of the S135L and Q188R (highly prevalent in white galactosemic children) missense mutations. The S135L mutation was found in 9 of 16 alleles of black children but not in white children; the Q188R mutation was found in 10 of 14 alleles examined in white galactosemic children and in 4 of 16 alleles in black galactosemic children. The GALT activity was near zero in the erythrocytes of white and black galactosemic children (0.26 +/- 0.28 vs 0.33 +/- 0.25 mumol/hr per gram of hemoglobin, respectively; p = 0.61) (normal 17 to 26 mumol/hr per gram), and no correlation of erythrocyte activity with genotype was observed. The GALT activity was higher in the leukocytes of black galactosemic children compared with white children (5 +/- 6 vs 1 +/- 2 mumol/hr per gram, respectively) (normal 172 to 374 mumol/hr per gram), but the difference was not statistically significant (p = 0.11). Analysis by genotype revealed that the two S135L homozygotes had much more leukocyte activity (9 and 17 mumol/hr per gram) than Q188R homozygotes or than all non-S135L allelic genotypes. Compound heterozygotes (S135L/G) had intermediate activity. The GALT protein was not detectable by Western blot in the erythrocytes of either white or black galactosemic children, as determined by antibodies specific for both C- and N-terminal sequences. The GALT protein was undetectable in the leukocytes of white galactosemic children, but leukocytes from black galactosemic children with the S135L mutation contained reduced but readily detectable GALT protein. Erythrocyte galactose 1-phosphate levels were significantly lower in galactosemic children with an S135L mutant allele (1.1 +/- 0.2 gm/dl) compared with children who had other mutations (3.1 +/- 0.9 mg/dl; p = 0.0001). The correlation of protein content data with activity levels in the blood cells suggests that the S135L missense mutation affects the stability of GALT protein to produce a deficiency state.

Adolescent↗
PubMed1999

Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1.

Amino Acid Sequence↗
PubMed2001

Identification of a cDNA clone encoding for a galactose-binding lectin from peanut (Arachis hypogaea) seedling roots.

A cDNA clone obtained from developing peanut (Arachis hypogaea) seedling roots, when expressed in Escherichia coli and insect cells (Sf9) gave a 29 kDa subunit protein. The native recombinant protein agglutinates neuraminidase treated human erythrocytes and the agglutination is inhibited by galactose. Nucleotide sequence and predicted amino acid sequence analyses indicate that it is different from peanut seed (PNA and SGL) and nodule (NGLa and NGLb) galactose-binding lectins.

Amino Acid Sequence↗
PubMed1997

Synthesis of novel 6-amido-6-deoxy-l-galactose derivatives as sialyl Lewis X mimetics.

The synthesis and biological potency of several sialyl Lewis X (SLe(x)) mimetics is described. These mimics incorporate all of the critical functional groups present in SLe(x) necessary for binding to E-selectin. L-Galactose is used to mimic the naturally occurring L-fucose residue in SLe(x) due to the identical arrangement of the 2-, 3-, and 4-hydroxyl groups. Several synthetically and enzymatically prepared amino acids were used to mimic the D-galactose residue. Because of the variability incorporated in the synthesis of these amino acids the spatial requirements necessary for efficient binding were investigated. A carboxylate bearing side chain was introduced as a sialic acid mimic and the chain length was varied to maximize biological activity. By investigating the optimal arrangement of these two factors mimics were produced which were up twofold more active than SLe(x).

Amines↗
PubMed1993

Reversible folding of UDP-galactose-4-epimerase from yeast Kluyveromyces fragilis.

UDP-galactose-4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule with one molecule of cofactor NAD per dimer. In presence of 8 M urea, the enzyme is inactivated with complete disorganization of its structure and dissociation of the subunits together with the cofactor. Dilution of the denaturant by sodium phosphate buffer (20 mM, pH 7.0) containing 1 mM NAD recovers the activity to the extent of 80-100%. At a monomer concentration of 0.8 microM, the reactivation follows second-order kinetics, k = 1.48 x 10(3) M-1 s-1, at 20 degrees C. The renatured enzyme resembles the native enzyme in terms of most of its physicochemical properties, e.g., circular dichroism spectrum, fluorescence spectrum, interaction with hydrophobic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS), hydrodynamic volume, Km for the substrate UDP-galactose, etc. The reactivation process has an energy of activation of 15.2 kcal/mol. The folding pathway could be divided into four major parts: in the first phase (within 10 min), the secondary and tertiary structures of the monomers are formed as found by circular dichroism and fluorescence spectroscopy; in the second phase (within 20 min), the folded monomers associate to form an inactive dimer as observed by size-exclusion HPLC; in the third phase, the NAD binding site is formed, which is possibly induced by the cofactor; and finally, NAD assembles over the cofactor binding site to yield an active holoenzyme (within 120 min). The third and fourth steps have been detected kinetically. They are slow and rate-limiting, depending upon the concentration of extraneous NAD.

Circular Dichroism↗
PubMed1991

19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change.

The Escherichia coli D-galactose and D-glucose receptor possesses a Ca(II)-binding site closely related in structure and metal-binding characteristics to the eukaryotic EF-hand sites. Only the structure of the Ca(II)-occupied site is known. To investigate the structural change triggered by Ca(II) and Sr(II) binding, we have used 19F NMR to probe five 5-fluorotryptophan (5F-Trp) and seven 3-fluorophenylalanine (3F-Phe) positions in the structure, extending the approach described in the preceding article. Of particular interest were two 5F-Trp residues near the N terminus of the Ca(II) site at positions 127 and 133. Substitution of the larger Sr(II) for Ca(II) triggered 19F NMR frequency shifts of the 5F-Trp127 and -133 resonances, indicating a detectable structural change in the Ca(II) site. In contrast, the three 5F-Trp resonances from distant regions of the structure exhibited no detectable frequency shifts. When the metal was removed from the Ca(II) site, the 5F-Trp127 and -133 frequencies shifted to a new value similar to that observed for free 5F-Trp in aqueous solvent, and this new frequency was a function of the H2O to D2O ratio, indicating that the residues had become solvent exposed. Metal removal yielded small or undetectable frequency shifts for the three distant 5F-Trp resonances and for four of the five resolved 3F-Phe resonances. The allosteric coupling of the metal and sugar binding sites was observed to be slight: depletion of metal ions was observed to reduce the D-galactose affinity of the receptor by 2-fold. Together the results indicate that the structural changes in the Ca(II) site are primarily localized in the region of the site. Removal of the metal ion from the site exposes the nearby 5F-Trp127 and -133 residues to the solvent, suggesting that the empty site has a more open structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1979

Nucleophile in the active site of Escherichia coli galactose-1-phosphate uridylyltransferase: degradation of the uridylyl-enzyme intermediate to N3-phosphohistidine.

The [32P]uridylyl-enzyme intermediate form of Escherichia coli galactose-1-P uridylyltransferase can be converted to a [32P]phosphoryl-enzyme by first cleaving the ribosyl ring with NaIO4 and then heating at pH 10.5 and 50 degrees C for 1 h. After alkaline hydrolysis of the [32P]phosphoryl-enzyme the major radioactive product is N3-[32P]phosphohistidine. A lesser amount of 32Pi is also produced as a side product of the hydrolysis of N3-[32P]phosphohistidine. No N1-phosphohistidine, N-phospholysine, or phosphoarginine can be detected in these hydrolysates. It is concluded that the nucleophile in galactose-1-P uridylyltransferase to which the uridylyl group is bonded in the uridylyl-enzyme intermediate is imidazole N3 of a histidine residue. This degradation procedure should have general applicability in the degradation and characterization of nucleotidyl-proteins.

Chemical Phenomena↗
PubMed1979

Uridine diphosphate galactose 4-epimerase. Alkylation of enzyme-bound diphosphopyridine nucleotide by p-(bromoacetamido)phenyl uridyl pyrophosphate, an active-site-directed irreversible inhibitor.

When UDP-galactose 4-epimerase is inactivated by p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP), the diphosphopyridine nucleotide (DPN) associated with this enzyme as a tightly bound coenzyme cannot be reduced by substrates or by UMP-activated reduction by glucose. Upon acid denaturation of the inactivated enzyme, the DPN released corresponded to 15-30% of that released from the native enzyme. When the enzyme is inactivated by [14C]BUP, about 80% of the radioactivity bound at the active site is released from the protein upon acid denaturation. When epimerase-[3H]DPN is inactivated with [14C]BUP, the 3H and 14C released from the protein upon denaturation of the complex cochromatograph on DEAE-Sephadex. Experiments with [nicotinamide-4-3H]DPN and [adenine-2,8-3H]DPN show that it is the adenine ring that is alkylated. The data suggest that the adenine ring of DPN in epimerase-DPN may be oriented near the glycosyl-binding subsite of this enzyme. Since the nicotinamide ring must also be near this site, it appears that the DPN may not be in an extended conformation when it is bound at the active site of UDP-galactose 4-epimerase from Escherichia coli.

Alkylation↗
PubMed2001

13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides.

UDP-galactose 4-epimerase contains the coenzyme NAD+ bound tightly at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structure and spectrophotometric data indicate that NAD+ may engage in electrostatic interactions with amino acid side chains that may regulate the reactivity of NAD+. In this work, we carried out NMR studies of [nicotinamide-4-13C]NAD+ bound to wild-type epimerase and epimerases mutated at amino acid residues in contact with NAD+. The 4-13C NMR chemical shifts revealed the following: The 4-13C chemical shift in wild-type epimerase is 149.9 ppm; mutation of Ser 124 to Ala changes it slightly by 0.2 ppm to 150.1 ppm; mutation of Tyr 149 to Phe results in a downfield perturbation of 2.7 ppm to 152.6 ppm; and the simultaneous mutation of Ser 124 to Ala and Tyr 149 to Phe also causes a downfield perturbation of 2.8 ppm to 152.7 ppm. Mutation of Lys 153 to Met results in a 13C chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild type and 1.8 ppm upfield from that of Y149F-epimerase. The 13C chemical shifts of nicotinamide C4 of NAD+ in these epimerases are correlated with their respective reactivities with NaBH3CN. In addition, reactivity of NAD+ in wild-type and S124A-epimerases displays pH dependence, with higher rates at lower pH where Tyr 149 in these two enzymes is protonated. The results support an electrostatic model in which repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD+, while the phenolate of Tyr 149 opposes the positive electrostatic field and attenuates the reactivity of NAD+. Ser 124 has very little effect on the electron distribution within the nicotinamide ring or the reactivity of NAD+. The effects of binding the substrate analogue P1-uridyl-P2-methyl diphosphate (Me-UDP) on the 4-13C chemical shifts are opposite to those induced by the mutations. MeUDP perturbs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-epimerases but has little or no effect in the cases of Y149F- or K153M-epimerases. The results support the postulate that NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr 149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys 153 and nicotinamide N1 of NAD+.

Borohydrides↗
PubMed1996

Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli.

UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose through a mechanism involving the transient reduction of NAD+. Here we describe the X-ray structures for epimerase complexed with NADH/UDP, and NAD+/UDP, refined to 1.8 and 2.0 angstrom, respectively. The alpha-carbon positions for the two forms of the enzyme are superimposed with a root-mean-square deviation of 0.36 A. Overall, the models for the reduced and oxidized proteins are very similar except for the positions of several side chains including Phe 178 and Phe 218. The most striking difference between the oxidized and reduced enzymes is the conformation of the nicotinamide ring of the dinucleotide. In the reduced protein, the nicotinamide ring adopts the anti conformation while in the oxidized enzyme the syn conformation is observed. There are also significant structural differences in UDP binding between the oxidized and reduced forms of the protein which most likely explain the observation that uridine nucleotides bind more tightly to epimerase/NADH than to epimerase/NAD+. Both van der Waals and electrostatic interactions between epimerase and NAD+ are extensive with 35 contacts below 3.2 angstrom as would be expected for enzyme that binds the dinucleotide irreversibly. This is in sharp contrast to the patterns typically observed for the NAD+-dependent dehydrogenases which bind nucleotides in a reversible fashion. While it has been postulated that the active site of epimerase must contain a base, the only potential candidates within approximately 5 A of both the NAD+ and the UDP are Asp 31, Asp 58, and ASP 295. These amino acid residues, however, are intimately involved in nucleotide binding and most likely do not play a role in the actual catalytic mechanism. Thus it may be speculated that an amino acid residue, other than glutamate, aspartate, or histidine, may be functioning as the active site base.

Binding Sites↗

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