Specificity and multiple forms of beta-galactosidase in the rat.
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ABSTRACT:
SUBMITTER: Furth AJ
PROVIDER: S-EPMC1264543 | biostudies-other | 1965 Oct
REPOSITORIES: biostudies-other
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Project description:The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5-8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)(8) TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.
| S-EPMC3391139 | biostudies-literature
Project description:Microbial ?-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMOS. Since this structural complexity is considered as the basis for the multiple biological functions of hMOS, it is important to broaden the variety of GOS structures. In this study, residue R484 near +1 subsite of the C-terminally truncated ?-galactosidase from Bacillus circulans (BgaD-D) was subjected to site saturation mutagenesis. Especially the R484S and R484H mutant enzymes displayed significantly altered enzyme specificity, leading to a new type of GOS mixture with altered structures and linkage types. The GOS mixtures produced by these mutant enzymes contained 14 structures that were not present in the wild-type enzyme GOS mixture; 10 of these are completely new structures. The GOS produced by these mutant enzymes contained a combination of (?1 ? 3) and (?1 ? 4) linkages, while the wild-type enzyme has a clear preference toward (?1 ? 4) linkages. The yield of the trisaccharide ?-d-Galp-(1 ? 3)-?-d-Galp-(1 ? 4)-d-Glcp produced by mutants R484S and R484H increased 50 times compared to that of the wild-type enzyme. These results indicate that residue R484 is crucial for the linkage specificity of BgaD-D. This is the first study showing that ?-galactosidase enzyme engineering results in an altered GOS linkage specificity and product mixture. The more diverse GOS mixtures produced by these engineered enzymes may find industrial applications.
| S-EPMC5330655 | biostudies-literature
Project description:1. The p-nitrophenyl beta-D-galactosidase asctivity in rat liver homogenates of lysosome-rich fractions was shown to be markedly affected by the ionic composition of the medium. A stimulation of the reaction rate at pH 5 was produced by most of the salts tested, which contained anions such as acetate, SO4(2-) and Cl-, and cations such as Na+, K= and Mg2+. The most pronounced effect was observed with MgCl2. Only potassium glutamate was inhibitory. 2. Five peaks of beta-galactosidase activity obtained by DEAE-cellulose chromatography were equally sensitive to changes in the ionic composition of the medium. In the presence of added NaC1, the whole rate-pH curve was displaced towards higher pH values, the optimum being shifted from 2.0-2.5 to 3.5. The stimulation at pH 5.0 appeared to be mainly due to changes in Vmax., whereas the apparent Km was slightly modified. 3. Unlike the total, the free beta-galactosidase activity remained unchanged or even declined when KC1 was added to the reaction medium.
| S-EPMC1172404 | biostudies-other
Project description:GM1 ganglioside beta-galactosidase (beta-Gal) is deficient in the autosomal recessive disorder GM1 gangliosidosis. A portion of the enzyme occurs in a complex with neuraminidase and an additional glycoprotein, protective protein, but the nature of the interactions conferring the stability of the complex is unknown. Affinity chromatography of beta-Gal on p-aminophenylthiogalactose-Sepharose (PATG-Sepharose) at pH 4.3, the pH optimum of beta-Gal, resulted in a 260-fold enrichment of beta-Gal, but the major protein in the fraction had an M(r) value of 74,000. Affinity chromatography on PATG-Sepharose at pH 5.2 showed substantial enrichment (4000-fold) of beta-Gal, and the mature form of the enzyme (M(r) 64,000) was the major protein in the preparation. Using h.p.l.c. molecular-sieve chromatography, we found that about 15% of the total beta-Gal occurred in a high-M(r) form (greater than 600,000), the presumptive complex, with 85% eluting at M(r) 150,000, suggestive of a dimer. This distribution was independent of both high (60 mg/ml) and low (5 mg/ml) protein concentration and the pH (pH 4.3 or 5.2) of the sample applied to the column. Furthermore, incubation for 90 min at 37 degrees C, conditions which had previously been suggested as optimal for formation of the complex, had no effect on this distribution. Further fractionation by anion-exchange chromatography and a second affinity column step yielded a beta-Gal preparation that contained a single polypeptide chain (M(r) 64,000), was devoid of neuraminidase and protective protein (absent carboxypeptidase activity), and when injected into rabbits gave rise to monospecific rabbit antisera. We conclude that the protein composition of the complex is variable (i.e. it is different when isolated at pH 4.3 and 5.2) and that the amount of beta-Gal tightly associated with the complex constitutes a small fraction of the total beta-Gal activity. The more prevalent form of the enzyme is a beta-Gal homodimer that is stable and devoid of either neuraminidase activity or protective protein.
| S-EPMC1132870 | biostudies-other
Project description:1. The specificity of purified sweet-almond alpha-galactosidase has been investigated with 17 substrates. 2. Some of them exhibited inhibition at high substrate concentrations but others did not. Both substrate types were bound and hydrolysed at the same site on the enzyme. 3. The enzyme is specific for alpha-d-galactosides and beta-l-arabinosides. It did not hydrolyse beta-d-galactosides or alpha-d-glucosides. 4. Among galactosides the order of decreasing rates of enzymic hydrolysis was: aryl alpha-galactosides; sugars; alkyl alpha-galactosides. 5. All substituents in the aryl moiety of aryl alpha-galactosides enhanced V(max.), the electron-releasing (-sigma) groups being more effective than the electron-withdrawing (+sigma) groups. The substituent groups did not alter K(m) appreciably. 6. Implications of these results are discussed from a mechanistic viewpoint.
| S-EPMC1270477 | biostudies-other
Project description:A gene encoding a third alpha-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other alpha-galactosidases revealed that it belongs to a subfamily of alpha-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic alpha-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger beta-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.
| S-EPMC91361 | biostudies-literature
Project description:1. The activities of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7.0 indicated slow- and fast-moving components of rat-kidney beta-galactosidase. 3. The fast-moving component is also associated with the total beta-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the beta-glucosidase component is a small acidic molecule, of molecular weight approx. 40000-50000, with optimum pH5.5-6.0 for beta-galactosidase and beta-glucosidase activities. 5. The major beta-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3.7.
| S-EPMC1270276 | biostudies-other
Project description:β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the β-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris.
| S-EPMC10607238 | biostudies-literature
Project description:The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.
| S-EPMC94720 | biostudies-literature
Project description:Binding of a single-chain Fv antibody to Escherichia coli β-galactosidase (β-gal) is known to stabilize the enzyme and activate several inactive point mutants, historically called antibody-mediated enzyme formation mutants. To understand the nature of this activation, we have determined by electron cryo-microscopy the structure of the complex between β-gal and the antibody scFv13R4. Our structure localizes the scFv13R4 binding site to the crevice between domains 1 and 3 in each β-gal subunit. The mutations that scFv13R4 counteracts are located between the antibody binding site and the active site of β-gal, at one end of the TIM-barrel that forms domain 3 where the substrate lactose is hydrolyzed. The mode of binding suggests how scFv stabilizes both the active site of β-gal and the tetrameric state.
| S-EPMC3988998 | biostudies-literature