Tryptic Digestion of Native Small‐Intestinal Sucrase · Isomaltase Complex: Isolation of the Sucrase Subunit

We here report the first molecular characterization of an ␣-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80°C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to ␣-glucosidases, ␣-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a ␤-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The ␣-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90°C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the ␣-xylosidase and the ␤-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.

Section: Discussionmentioning

confidence: 99%

Identification and Molecular Characterization of the First α-Xylosidase from an Archaeon

Moracci¹,

Cobucci‐Ponzano²,

Trincone³

et al. 2000

“…This type of catalytic site (WIDMNE) has an aspartic acid (D) catalytic site (45,46). This catalytic site is conserved in other carbohydrate hydrolases including human, rabbit, and rat SIM (29, 30, 47); mouse and human (36, 48) lysosomal maltase; fungal maltases from Aspergillus niger, Mucor javanicus, Schwanniomyces occidentalis (49, 50, 51); and plant maltase from sugar beets and barley (52,53).…”

Section: Downloaded Frommentioning

confidence: 99%

“…This type of catalytic site (WIDMNE) has an aspartic acid (D) catalytic site (45,46 and plant maltase from sugar beets and barley (52,53).…”

Section: Figmentioning

confidence: 99%

Human Small Intestinal Maltase-glucoamylase cDNA Cloning

Nichols¹,

Eldering²,

Avery³

et al. 1998

Self Cite

112

It has been hypothesized that human mucosal glucoamylase (EC 3.2.1.20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal ␣-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltaseglucoamylase was purified by immunoisolation and partially sequenced. Maltase-glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.Starches are a mixture of two structurally different polysaccharides: amylose, a linear [4-O-␣-D-glucopyranosyl-D-glucose] n polymer, and amylopectin, with additional 6-O-␣-D-glucopyranosyl-D-glucose links (about 4% of total), which result in a branched configuration. Dietary starches are a mixture of approximately 25% amylose in amylopectin, a fact of nutritional significance because of the multienzyme complexity of the mammalian starch digestion pathway (1). ␣-amylase (EC 3.2.1.1) is the endoenzyme found in mature human salivary and pancreatic secretions that produces linear maltose oligosaccharides by hydrolysis of ␣134 linkages of amylose (2, 3). ␣-amylase bypasses the ␣1 3 6 linkages of amylopectin and produces branched isomaltose oligosaccharides. The starchderived oligosaccharides are not fermentable by yeast without further processing by ␤-amylase (EC 3.1.1.2), which hydrolyzes the nonreducing ends at 134 and 136 linkages (2). In mammals, hydrolysis of the nonreducing ends is carried out by small intestinal mucosal brush border-anchored sucrase-isomaltase (SIM) 1 (EC 3.2.1.48 and 3.2.1.10) and maltase-glucoamylase (MGA) (EC 3.2.1.20 and 3.2.1.3) complexes (1). Enzyme substrate specificities of SIM overlap with those of MGA. In vivo, SIM accounts for 80% of maltase (1,4-O-␣-D-glucanohydrolase) activity, all sucrase (D-glucopyranosyl-␤-D-fructohydrolase) activity, and almost all isomaltase (1, 6-O-␣-D-glucanohydrolase) activity (1). MGA accounts for all glucoamylase exoenzyme (1,4-O-␣-D-glucanohydrolase) activity for amylose and amylopectin substrates, 1% of isomaltase activity, and 20% of maltase activity (1...

“…For example, an aspartate that functions as a catalytic nucleophile in a sucrose, ␣-glucan glycosyltransferase, has been identified in Streptococcus ␣-glucosyltransferase by denaturation trapping using radiolabeled sucrose (9). Similarly, in a second group of ␣-glycosyl hydrolases (Family 31), Asp-505 and Asp-1394 have been identified as active site residues in each of the two homologous active sites in the sucraseisomaltase complex by affinity labeling with conduritol B epoxide and have been suggested to be the catalytic nucleophiles (10). The asparagine mutant of the equivalent Asp-518 in human lysosomal ␣-glucosidase exhibits an estimated 6% of wildtype activity, indicating that this residue likely plays an important role in catalysis (11), although this 16-fold reduction in activity is much less than the 10 5 -fold reduction observed upon analysis of nucleophile mutants in mechanistically similar ␤-glycosidases (12)(13)(14), leaving some doubt concerning the role of Asp-518.…”

mentioning

confidence: 99%

Unequivocal Identification of Asp-214 as the Catalytic Nucleophile of Saccharomyces cerevisiae α-Glucosidase Using 5-Fluoro Glycosyl Fluorides

McCarter

Withers²

1996

130

Yeast ␣-glucosidase is a member of a sequence-related family of ␣-glycosidases (Family 13) that includes important digestive ␣-amylases and ␣-glucosidases. These enzymes catalyze the hydrolysis of ␣-linked oligosaccharides by a two-step mechanism involving a glycosylenzyme intermediate. This Glycosyl hydrolases of Family 13 (the ␣-amylase family (1, 2)), which include ␣-amylases and some ␣-glucosidases and oligo-1,6-glucosidases, are important enzymes involved in the digestion of starch and other ␣-linked oligosaccharides in bacteria, plants, and animals. These glycosyl hydrolases cleave the glycosidic bond with net retention of configuration, thus are retaining enzymes that function via a two-step, double displacement mechanism. The first step involves the attack of an enzymic nucleophile at the anomeric center of the sugar, along with general acid catalysis to facilitate loss of the leaving group, leading to the formation of a ␤-D-glycosyl enzyme intermediate. This intermediate can then undergo hydrolysis in a second step, with general base catalysis to facilitate attack of water, resulting in cleavage of the glycosidic bond with net retention of anomeric configuration (see Scheme 2). The active site of these enzymes is known to include a trio of conserved carboxylic acids (3-7). Presumably, one residue functions as the catalytic nucleophile, one as a general acid/base, and the third possibly affords additional stabilization of developing positive charge or modulates the ionization behavior of the other catalytic residues.Considerable progress has been made toward elucidating the roles of these carboxylates in Family 13 enzymes, involving kinetic analyses of site-specific mutants (Ref. 8 and references therein) and x-ray crystallography (3-6). Labeling studies have also been useful. For example, an aspartate that functions as a catalytic nucleophile in a sucrose, ␣-glucan glycosyltransferase, has been identified in Streptococcus ␣-glucosyltransferase by denaturation trapping using radiolabeled sucrose (9). Similarly, in a second group of ␣-glycosyl hydrolases (Family 31), Asp-505 and Asp-1394 have been identified as active site residues in each of the two homologous active sites in the sucraseisomaltase complex by affinity labeling with conduritol B epoxide and have been suggested to be the catalytic nucleophiles (10). The asparagine mutant of the equivalent Asp-518 in human lysosomal ␣-glucosidase exhibits an estimated 6% of wildtype activity, indicating that this residue likely plays an important role in catalysis (11), although this 16-fold reduction in activity is much less than the 10 5 -fold reduction observed upon analysis of nucleophile mutants in mechanistically similar ␤-glycosidases (12-14), leaving some doubt concerning the role of Asp-518. This activity was not, however, based on rigorously purified enzyme, and the assay method used in this study could not distinguish this level of activity from background. Furthermore, lysosomal ␣-glucosidase and sucrase-isomaltase (Family 31) and the ␣-amylases ...