Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley ␣-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley ␣-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely ؊7 through ؉2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley ␣-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1 D180A ) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."

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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Background:The details of the catalytic mechanism of cellobiose 2-epimerase (CE) remains unclear. Results: The crystal structures of Rhodothermus marinus CE in the apo form and complexes with its substrates/products 4-O-␤-D-glucopyranosyl-D-mannnose, epilactose, or cellobiitol (reaction intermediate analog) were elucidated. Conclusion: Epimerization catalyzed by CE proceeds through ring opening, deprotonation/reprotonation, carbon-carbon bond rotation, and ring closure. Significance: This study yielded structural insights into epimerization catalyzed by CE.

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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.

Enzymatic properties of barley a-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites 25/26 (Cys95 ! Ala/Thr) and þ1/þ 2 (Met298 ! Ala/ Asn/Ser) as well as in the double mutants, Cys95 ! Ala/ Met298 ! Ala/Asn/Ser. Cys95 ! Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k cat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl b-D-maltoheptaoside (Cl-PNPG 7 ), respectively, but fivefold to 20-fold higher K m . The Cys95 ! Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 ! Ala and wild-type. Met298 ! Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k cat and k cat /K m for Cl-PNPG 7 are # 30% and # 10% of wild-type, respectively. The activity of Cys95 ! Ala/Met298 ! Ala/ Asn/Ser is 100-180% towards starch, and the k cat /K m is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG 7 , respectively, emphasizing the strong impact of the Cys95 ! Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG 7 and amylose/Cl-PNPG 7 are 2.8-to 270-fold and 1.2-to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites 25 and þ2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites þ1 and þ2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6 000 -maltotriosyl-maltohexaose thus covering subsites 2 1 to þ5, while productive binding of unbranched substrate involves subsites 2 3 to þ3.Keywords: glycoside hydrolase family 13; (b/a) 8 barrel; site-directed mutagenesis; substrate specificity; branched malto-oligosaccharide.a-Amylase (a-1,4-D-glucan glucanohydrolase, EC 3.2.1.1) catalyzes hydrolysis of internal a-1,4-glucosidic linkages in starch and related saccharides [1]. a-Amylases belong to glycoside hydrolase family 13, which includes enzymes exhibiting 26 different specificities, e.g. a-glucosidase, cyclomaltodextrinase, pullulanase, cyclodextrin glucanotransferase, branching enzyme, and amylosucrase and showing very low sequence similarity [2,3]. In spite of a considerable amount of structural information available, there is a strong need to understand the impact of enzyme -substrate interactions at the level of specific binding subsites for the ultimate exploitation in rational design of enzymes with desired properties. The specificity of a family 13 member has been engineered into another enzyme class in only a few cases [4].Three-dimensional structures have been solved for several family members and have been shown to share a catalytic (b/a) 8 barrel (domain A), a domain that protrudes between the third b strand and a he...

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