Identification and Characterization of Two Novel Alpha-D-Galactosidases from Pedobacter heparinus.

Two putative α-D-galactosidases (α-GALs) belonging to glycosyl hydrolase family 27, and originating from the rather unexplored bacterial strain Pedobacter heparinus, were cloned and biochemically characterized. The recombinant enzymes designated as PhAGal729 and PhAGal2920 showed comparable biochemical properties: the optimum pH values were determined to be pH 5.0 and 5.5, and temperature optima lay between 30°C and 37°C, respectively. Both α-GALs were not dependent on the presence of divalent metal ions, and the addition of EDTA had no influence on enzymatic activity. The activity of both enzymes substantially increased in the presence of Fe3(+) ions. Both enzymes were inhibited by sodium dodecyl sulfate (SDS) and urea. α-GALs from P. heparinus were highly specific in hydrolyzing glycosides with α-1,2/3/4 or α-1,6-linked galactose to other sugars, whereas other glycosides such as α-linked N-acetylgalactosamine, N-acetylglucosamine or glucose residues were not released. Nevertheless, neither PhAGal729 nor PhAGal2920 were able to remove α-linked galactose epitopes from native human erythrocytes. The facile expression and purification procedures in combination with wide substrate specificities make α-GALs from P. heparinus potential candidates for applications in analytical research, and food- and biotechnology.

[Structural analysis of genes participating in melibiose fermentation and isocitrate lyase production in Yersinia pestis strains of main and non main subspecies].

Comparative analysis of nucleotide sequences of genes participating in melibiose fermentation and isocitrate lyase production was conducted in 90 natural Yersinia pestis strains of main and non main subspecies. It was ascertained that the lack of the ability to utilize disaccharide melibiose in strains of the main subspecies is caused by integration of the insertion sequence IS285 at 73 bp from the beginning of the structural gene melB that encodes the transport protein galactoside permease. In contrast, strains of non main subspecies (caucasica, altaica, and ulegeica) contain the intact gene melB and are capable of fermenting melibiose. Differences in the manifestation of the other differential trait, production of isocitrate lyase, are connected with the presence of mutation (insertion of two nucleotides +CC) in the regulatory gene iclR encoding repressor protein of the acetate operon, which is the reason for constitutive synthesis of this enzyme. Strains of non main subspecies do not contain mutations in gene iclR, and this correlates in these strains with their capacity for inducible synthesis of isocitrate lyase.

Evolution and biochemistry of family 4 glycosidases: implications for assigning enzyme function in sequence annotations.

Glycosyl hydrolase Family 4 (GH4) is exceptional among the 114 families in this enzyme superfamily. Members of GH4 exhibit unusual cofactor requirements for activity, and an essential cysteine residue is present at the active site. Of greatest significance is the fact that members of GH4 employ a unique catalytic mechanism for cleavage of the glycosidic bond. By phylogenetic analysis, and from available substrate specificities, we have assigned a majority of the enzymes of GH4 to five subgroups. Our classification revealed an unexpected relationship between substrate specificity and the presence, in each subgroup, of a motif of four amino acids that includes the active-site Cys residue: alpha-glucosidase, CHE(I/V); alpha-galactosidase, CHSV; alpha-glucuronidase, CHGx; 6-phospho-alpha-glucosidase, CDMP; and 6-phospho-beta-glucosidase, CN(V/I)P. The question arises: Does the presence of a particular motif sufficiently predict the catalytic function of an unassigned GH4 protein? To test this hypothesis, we have purified and characterized the alpha-glucoside-specific GH4 enzyme (PalH) from the phytopathogen, Erwinia rhapontici. The CHEI motif in this protein has been changed by site-directed mutagenesis, and the effects upon substrate specificity have been determined. The change to CHSV caused the loss of all alpha-glucosidase activity, but the mutant protein exhibited none of the anticipated alpha-galactosidase activity. The Cys-containing motif may be suggestive of enzyme specificity, but phylogenetic placement is required for confidence in that specificity. The Acholeplasma laidlawii GH4 protein is phylogenetically a phospho-beta-glucosidase but has a unique SSSP motif. Lacking the initial Cys in that motif it cannot hydrolyze glycosides by the normal GH4 mechanism because the Cys is required to position the metal ion for hydrolysis, nor can it use the more common single or double-displacement mechanism of Koshland. Several considerations suggest that the protein has acquired a new function as the consequence of positive selection. This study emphasizes the importance of automatic annotation systems that by integrating phylogenetic analysis, functional motifs, and bioinformatics data, may lead to innovative experiments that further our understanding of biological systems.

Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen.

In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.

A potent bicyclic inhibitor of a family 27 alpha-galactosidase.

Two isomeric bicyclo[4.1.0]heptane analogues of the glycosidase inhibitor galacto-validamine, (1R*,2S,3S,4S,5S,6S*)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptane-2,3,4-triol, have been synthesized in 13 steps from 2,3,4,6-tetra-O-benzyl-D-galactose. The inhibitory activities of the two conformationally restricted amines, and their corresponding acetamides, were measured against commercial alpha-galactosidase enzymes from coffee bean and E. coli. The activity of the glycosyl hydrolase family GH27 enzyme (coffee bean) was competitively inhibited by the 1R,6S-amine (7), a binding interaction that was characterized by a K(i) value of 0.541 microM. The GH36 E. coli alpha-galactosidase exhibited a much weaker binding interaction with the 1R,6S-amine (IC(50)= 80 microM). The diastereomeric 1S,6R-amine (9) bound weakly to both galactosidases, (coffee bean, IC(50)= 286 microM) and (E. coli, IC(50)= 2.46 mM).

Purification and characterization of alpha-galactosidase from sunflower seeds.

From 100 g sunflower seeds, 1.2 mg purified alpha-galactosidase was obtained with an overall yield of 51%. The alpha-galactosidase acted on both terminal alpha-galactosyl residues and side-chain alpha-galactosyl residues of the galactomanno-oligosaccharides and galactomannans. The cDNA coding for sunflower alpha-galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40,263. Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence. The deduced amino acid sequences of mature enzyme and alpha-galactosidases from coffee, guar and Mortierella vinacea alpha-galactosidase II showed over 81%, 77%, and 47% homology, respectively. These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups.

The cloning and characterization of alpha-galactosidase present during and following germination of tomato (Lycopersicon esculentum Mill.) seed.

alpha-Galactosidase (EC 3.2.1.22) is present in the embryo, micropylar and lateral endosperm of seeds of tomato during and following germination. Its activity is unchanged even when germination of the seeds is prevented by an osmoticum. It is also present in the developing and mature dry seed. A cDNA clone for tomato seed alpha-galactosidase (LeaGal) has been isolated and the characteristics of the protein deduced; the predicted molecular mass of the mature enzyme is 39.8 kDa, with a pI of 4.91. The tomato alpha-galactosidase has a high homology (>62%) at the amino acid level with that of other plant alpha-galactosidases. A hydrophobic signal peptide region is identified which is indicative that the enzyme enters the lumen of the endoplasmic reticulum during its translation, prior to its export to the protein body or cell wall, the presumed sites of its substrates. Using amino acid alignment and phylogenetic analysis, key amino acids have been identified, and relationships to other alpha-galactosidases inferred. Southern hybridization analyses show that the enzyme is derived from a single gene (for which a partial sequence has been obtained) and yet there are at least three different isoforms within the seed; post-translational modifications are thus presumed to occur. From Northern hybridization studies it is evident that alpha-galactosidase transcripts are present in the lateral and micropylar endosperm during and following germination, and also to a lesser extent in the embryo.

Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase.

Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase clan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4', 6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2, 2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2, 6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.