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PubMed2006

Purification, identification and molecular cloning of glycoside hydrolase family 15 glucoamylase from the brown-rot basidiomycete Fomitopsis palustris.

The brown-rot basidiomycete Fomitopsis palustris produces a major extracellular enzyme of 72 kDa when the fungus is incubated in cellulose culture with 0.2% cellobiose. This protein was purified by column chromatography, and the amino acid sequences of its proteolytic fragments were analyzed. The N-terminal amino acid sequence of one of the fragments showed high identity with fungal glycoside hydrolase family 15 glucoamylases. As its kinetic efficiency increased in proportion to the degree of polymerization of the substrate, the protein was identified as a glucoamylase. A cDNA encoding the glucoamylase (gla) was cloned by reverse transcriptase PCR.

Amino Acid Sequence↗
PubMed2006

Gene cloning and heterologous expression of glycoside hydrolase family 55 beta-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium.

The basidiomycete Phanerochaete chrysosporium produces several beta-1,3-glucanases when grown on laminarin, a beta-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown beta-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched beta-1,3/1,6-glucan as well as linear beta-1,3-glucan, the kinetic features of the enzyme were compared with those of other beta-1,3-glucanases.

Amino Acid Sequence↗
PubMed1995

Analogues of disaccharides and glycosides containing a cyclic guanidinium structure show varying inhibitory effects on glycoside hydrolases.

By condensation of 1,3-diamino-2,4-(R)-O-benzylidene-1,3-dideoxy-D-erythritol (3) and 1,3-diamino-2,4-di-O-benzyl-1,3-dideoxy-D-threitol (4) with methyl 2,3,6-tri-O-benzyl-4-deoxy-4-iso-thiocyanato-beta-D-glucopyranosid e (9) the (1-->4)-linked disaccharide analogues 4-deoxy-4-[(4R,5S)-5-hydroxy-4-(hydroxymethyl)-1,4,5,6-tetrahydropyri midin-2- yl[amino-alpha,beta-D-glucopyranose hydrochloride (15) and 4-deoxy-4-[(4R,5R)-5-hydroxy-4-(hydroxymethyl)-1,4,5,6-tetrahydropyri midin- 2-yl]amino-alpha,beta-D-glucopyranose hydrochloride (18) were synthesized. By the same reaction sequence, using 3 and methyl isothiocyanate, the glycoside analogue (4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-methylamino-1,4,5,6- tetrahydropyrimidine hydrochloride (20) was obtained. All compounds possess in their 'glyconic' moiety the flat guanidinium group, mimicking a glucopyranosyl cation. Together with the previously synthesized (1-->6)-linked disaccharide analogues 6-deoxy-6-[(4R,5S)-5-hydroxy-4-(hydroxymethyl)-1,4,5,6- tetrahydropyrimidin-2-yl]amino-alpha,beta-D-glucopyranose hydrochloride (1) and 6-deoxy-6-[(4R,5R)-5-hydroxy-4-(hydroxy-methyl)-1,4,5,6- tetrahydropyrimidin-2-yl]amino-alpha,beta-D-glucopyranose hydrochloride (2), a possible inhibitory effect on the action of alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, and beta-D-galactosidase was investigated. All compounds, except 20 with alpha-D-glucosidase where no inhibition could be detected, showed either competitive or mixed competitive inhibition with all enzymes. The effects of the disaccharide analogues were generally weaker as compared to the effect of the previously synthesized configurationally related nitrophenyl glycoside analogues (4R,5S)-5-hydroxy-4-(hydroxymethyl)-2-(p-nitrophenyl)amino-1,4,5,6- tetrahydropyrimidine hydrochloride (21) and (4R,5R)-5-hydroxy-4-(hydroxymethyl)-2-(p-nitrophenyl)amino-1,4,5,6- tetrahydropyrimidine hydrochloride (22). On the basis of experimental results, different binding hydrochloride (22). On the basis of experimental results, different binding modes of competitive inhibitors to the active site of corresponding enzymes are discussed.

Carbohydrate Conformation↗
PubMed2003

Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in glycoside hydrolase family 1.

The Sphingomonas paucimobilis beta-glucosidase Bgl1 is encoded by the bgl1 gene, associated with an 1308 bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bgl1 protein through the inner membrane to the periplasmic space. A His(6)-tagged 44.3 kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bgl1 gene lacking the region encoding the putative signal peptide. Mature beta-glucosidase Bgl1 is specific for aryl-beta-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bgl1 and other members of the glycoside hydrolase (GH) family 1 enzymes. At subsite -1, the conserved residues required for catalysis by GH1 enzymes are present in Bgl1 with only minor differences. Major differences are found at subsite +1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH1 enzymes, revealing an absence of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bgl1, the first characterized beta-glucosidase from the genus Sphingomonas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin.

Amino Acid Sequence↗
PubMed2005

Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum.

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

Amino Acid Sequence↗
PubMed2006

Molecular basis of substrate specificity in family 1 glycoside hydrolases.

ss-glycosidases are active upon a large range of substrates. Besides this, subtle changes in the substrate structure may result in large modifications on the ss-glycosidase activity. The characterization of the molecular basis of ss-glycosidases substrate preference may contribute to the comprehension of the enzymatic specificity, a fundamental property of biological systems. ss-glycosidases specificity for the monosaccharide of the substrate nonreducing end (glycone) is controlled by a hydrogen bond network involving at least 5 active site amino acid residues and 4 substrate hydroxyls. From these residues, a glutamate, which interacts with hydroxyls 4 and 6, seems to be a key element in the determination of the preference for fucosides, glucosides and galactosides. Apart from this, interactions with the hydroxyl 2 are essential to the ss-glycosidase activity. The active site residues forming these interactions and the pattern of the hydrogen bond network are conserved among all ss-glycosidases. The region of the ss-glycosidase active site that interacts with the moiety (called aglycone) which is bound to the glycone is formed by several subsites (1 to 3). However, the majority of the non-covalent interactions with the aglycone is concentrated in the first one, which presents a variable spatial structure and amino acid composition. This structural variability is in accordance with the high diversity of aglycones recognized by ss-glycosidases. Hydrophobic interactions and hydrogen bonds are formed with the aglycone, but the manner in which they control the ss-glycosidase specificity still remains to be determined.

Amino Acid Sequence↗
PubMed2003

The mechanisms by which family 10 glycoside hydrolases bind decorated substrates.

Endo-beta-1,4-xylanases (xylanases), which cleave beta-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the three-dimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the +1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the +4, +3, +1, and -3 subsites may allow accommodation of the alpha-1,2-linked 4-O-methyl-d-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the +1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3'-linked l-arabinofuranoside decorations is observed in the -2 subsite and could, most likely, be tolerated when bound to xylosides in -3 and +4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as alpha-glucuronidase. The data described in this report and in the accompanying paper indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases.

Bacterial Proteins↗