Magali Remaud-Siméon

Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes

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 × 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.

Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases.

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 α-1,3 and/or α-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.

Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

Molecular Basis of the Amylose-like Polymer Formation Catalyzed by Neisseria polysaccharea Amylosucrase

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with α-amylases. Exactly how this enzyme catalyzes the formation of α-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.

Role of the Two Catalytic Domains of DSR-E Dextransucrase and Their Involvement in the Formation of Highly α-1,2 Branched Dextran

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

Functional and Structural Characterization of alpha-(1 -> 2) Branching Sucrase Derived from DSR-E Glucansucrase

Background: The transglucosidase GBD-CD2 shows a unique α-(1→2) branching specificity among GH70 family members when catalyzing dextran glucosylation from sucrose. Results: The truncated form ΔN123-GBD-CD2 was biochemically studied and structurally characterized at 1.90 Å resolution. Conclusion: Dextran recognition and regiospecificity clearly involves a residue in subsite +1. Significance: This is the first three-dimensional structure of a GH70 enzyme that reveals determinants of α-(1→2) linkage specificity. ΔN123-glucan-binding domain-catalytic domain 2 (ΔN123-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN123-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN123-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN123-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN123-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.

Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression

The study of exopolysaccharide production by heterofermentative sourdough lactic acid bacteria has shown that Weissella strains isolated from sourdoughs produce linear dextrans containing α-(1→6) glucose residues with few α-(1→3) linkages from sucrose. In this study, several dextran-producing strains, Weissella cibaria and Weissella confusa, isolated from sourdough, were characterized according to carbohydrate fermentation, repetitive element-PCR fingerprinting using (GTG)(5) primers and glucansucrase activity (soluble or cell-associated). This study reports, for the first time, the characterization of dextransucrase from Weissella strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ polymer production (after incubation with sucrose) from enzymatic fractions harvested from both sucrose and glucose culture media. Results demonstrate that dextransucrase activity was mainly soluble and associated with a constitutive 180-kDa protein. In addition, microsequencing of the active dextransucrase from W. cibaria LBAE-K39 allowed the design of specific primers that could detect the presence of glucansucrase encoding genes similar to GTFKg3 of Lactobacillus fermentum Kg3 and to DSRWC of W. cibaria CMU. This study hence indicates that sourdough Weissella strains synthesize original dextransucrase.

Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so‐called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position‐restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009.

Design of α-Transglucosidases of Controlled Specificity for Programmed Chemoenzymatic Synthesis of Antigenic Oligosaccharides

Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-l-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-d-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chemoenzymatic pathway and enzyme engineering in glycochemistry.

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2‐substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild‐type BCL could be improved by chemical engineering of the substrates molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)‐2‐chloro ethyl 2‐bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15‐fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.