The human gut microbiome is a complex ecosystem composed mainly of uncultured bacteria. It plays an essential role in the catabolism of dietary fibers, the part of plant material in our diet that is not metabolized in the upper digestive tract, because the human genome does not encode adequate carbohydrate active enzymes (CAZymes). We describe a multi-step functionally based approach to guide the in-depth pyrosequencing of specific regions of the human gut metagenome encoding the CAZymes involved in dietary fiber breakdown. High-throughput functional screens were first applied to a library covering 5.4 3 10 9 bp of metagenomic DNA, allowing the isolation of 310 clones showing beta-glucanase, hemicellulase, galactanase, amylase, or pectinase activities. Based on the results of refined secondary screens, sequencing efforts were reduced to 0.84 Mb of nonredundant metagenomic DNA, corresponding to 26 clones that were particularly efficient for the degradation of raw plant polysaccharides. Seventy-three CAZymes from 35 different families were discovered. This corresponds to a fivefold target-gene enrichment compared to random sequencing of the human gut metagenome. Thirty-three of these CAZy encoding genes are highly homologous to prevalent genes found in the gut microbiome of at least 20 individuals for whose metagenomic data are available. Moreover, 18 multigenic clusters encoding complementary enzyme activities for plant cell wall degradation were also identified. Gene taxonomic assignment is consistent with horizontal gene transfer events in dominant gut species and provides new insights into the human gut functional trophic chain.

Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various ␣-1,3 and/or ␣-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases. Glucansucrases from Glycoside-Hydrolase (GH)2 -family 70 (EC. 2.4.1.5) are extracellular enzymes produced by lactic acid bacteria of the genus Leuconostoc, Streptococcus, or Lactobacillus (1). From sucrose, they catalyze the synthesis of high molecular weight glucans. They can also produce oligosaccharides or glucoconjugates by a transglucosylation reaction from the sucrose donor to an exogenous acceptor, and this so called "acceptor reaction" occurs at the cost of polymer synthesis (2, 3). An interesting diversity exists in the GH-family 70, where there are enzymes able to synthesize all the types of glucosidic linkages, namely ␣-1,2; ␣-1,3; ␣-1,4; or ␣-1,6 glucosidic bonds. So, depending on the enzyme specificity, a wide range of glucans can be produced, varying in terms of size, structure, degree of branches and spatial arrangements.Primary structures of at least 44 different glucansucrases are now available in GH-family 70.3 With an average predicted molecular mass of more than 160,000 Da, they all show the same organization consisting of a variable region at the N terminus, a conserved catalytic domain, and a C-terminal domain typically containing a series of homologous repeating units. In a number of streptococcal glucansucrases, as well as for the L. mesenteroides NRRL B-512F dextransucrase, the repeats have been demonstrated to play a role in enzyme glucan bindi...

The three-dimensional structure of A-amylose crystals, as a model of the crystal domains of A-starch granules, was revised using synchrotron radiation microdiffraction data collected from individual micron-sized single crystals. The resulting datasets allowed a determination of the structure with conventional X-ray structure determination techniques normally used for small molecules and not for polymers. Whereas the gross features of this improved structure do not differ extensively from previous structure determination, the high resolution of the diffraction diagrams, which is unusual for a crystalline polymer, allowed the resolution of important new fine details. These include a distortion of the amylose double helices resulting from the occurrence of two intracrystalline molecules of water and a tight network of hydrogen bonds involving each of the primary and secondary hydroxyl groups of the glucosyl moieties. Pairs of water molecules are located in discrete pockets that do not interfere with one another. In addition, the refinement of the new structure indicates a "parallel-down" organization of the amylose molecules within the unit cell as opposed to the previous "parallel-up" model. This new feature indicates that within the crystals, the nonreducing ends of the amylose molecules are oriented toward the c-axis direction of the unit cell. The description of this geometry is important to correlate the crystallography of the granules of A-starch with their ultrastructure and their mode of biosynthesis.Here, we present for the first time the resolution of the structure of a polymer crystal from a full X-ray dataset collected on micron-sized polymer single crystals using synchrotron radiation microdiffraction. This achievement is a substantial advance, which opens the way to many more studies since the technique of growing polymer and biopolymer single crystals is well established.3