SusB, an 84-kDa ␣-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6 Å resolution. SusB hydrolyzes the ␣-glucosidic linkage, with inversion of anomeric configuration liberating the ␤-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only ␣-1,4-glucosidic linkages but also ␣-1,6-, ␣-1,3-, and ␣-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu 532 is positioned to provide protonic assistance to leaving group departure, with Glu 439 and Glu 508 both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of ␣-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.

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BackgroundPropolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, Apis mellifera propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity.MethodsPropolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity in vitro against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G2), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy.ResultsThe crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC50 values across the five cancer cell lines ranging from 41.3 to 52.4 μg/ml and from 43.8 to 53.5 μg/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar in vitro antiproliferation/cytotoxicity IC50 values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 μg/ml for the cardanol and < 3.13 to 5.97 μg/ml (6.82 - 13.0 μM) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts.ConclusionThis is the first report that Thai A. mellifera propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.

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cDNA encoding Schizosaccharomyces pombe a-glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M r 106 138. The deduced amino-acid sequence showed a high homology to those of a-glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the a-glucosidase family II. By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the a-glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO 2 ) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.Keywords: a-glucosidase; proton donor in catalytic reaction; site-directed mutagenesis.a-Glucosidase (EC 3.2.1.20, a-d-glucoside glucohydrolase) catalyzes the liberation of a-glucose from nonreducing terminals of various glucoside substrates. Many a-glucosidases, which show broad substrate specificities [1], have been purified from mammals, plants and microorganisms. They are divided into two groups, a-glucosidase families I and II, based on their amino-acid sequences [2]; families I and II belong to families 13 and 31 of the glycoside hydrolases, respectively [3,4]. Family I, for example, Saccharomyces carlsbergensis (Saccharomyces cerevisiae) a-glucosidase [5] and Bacillus cereus a-glucosidase [6], has the four regions that are conserved among the amino-acid sequences of a-amylases [7]. On the other hand, enzymes such as human lysosomal [8], Aspergillus niger [9,10], and sugar beet [11] a-glucosidases, belong to a-glucosidase family II, and do not have the four conserved regions. There is no similarity in the overall primary structures of a-glucosidase and a-amylase, or of the a-glucosidase families I and II. The threedimensional structure of a-glucosidase was solved only in B. cereus [12], and the catalytic amino-acid residues were deduced from the structure of the catalytic regions conserved in a-amylase [13]. As for the a-glucosidases in family II, however, only one catalytic amino-acid residue was confirmed by chemical modification with conduritol B epoxide (1-d-1,2-anhydro-myo-inositol; CBE). CBE is a known mechanism-based inactivator of a-glucosidase [14±19] that binds specifically to the essential b-carboxyl group of the Asp residue at the active site, which is the catalytic nucleophile (-COO 2 ): the Asp505 and Asp1349 residues of rabbit intestinal sucrase±isomaltase complex [15,16], the Asp518 of human lysosomal a-glucosidase [17], the Asp469 of sugar beet a-glucosidase [18], and the Asp224 of the P2 subunit of A. niger a-glucosidase [19]. The previous kinetic and chemical modification analysis suggested that carboxyl...

Background:The origin of specificity of plant ␣-glucosidases for long malto-oligosaccharides remains uncertain. Results: The crystal structure and mutational analyses of sugar beet ␣-glucosidase revealed its substrate binding properties. Conclusion:The long-substrate specificity was described as two structural elements, the N-loop and subdomain b2. Significance: A slight structural difference leads to significant differences in specificity for varying chain lengths of substrate.

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Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.