In Vitro Synthesis and Crystallization of ß-1,4-Mannan.

In vitro polymerization of ß-mannans is a challenging reaction due to the steric hindrance confered by the configuration of mannosyl residues and the thermodynamic instability of the ß-anomer. Whatever the approach used to date-whether chemical, or enzymatic with glycosynthases and mannosyltransferases-pure ß-1,4-mannans have never been synthesized in vitro. This has limited attempts to investigate their role in the production of plant and algal cell walls, in which they are highly abundant. It has also impeded the exploitation of their properties as biosourced materials. In this paper, we demonstrate that TM1225, a thermoactive glycoside phosphorylase from the hyperthermophile species Thermotoga maritima, is a powerful biocatalytic tool for the ecofriendly synthesis of pure ß-1,4-mannan. The recombinant production of this enzyme and its biochemical characterization allowed us to prove that it catalyzes the reversible phosphorolysis of ß-1,4-mannosides, and determine its role in the metabolism of the algal mannans on which T. maritima feeds in submarine sediments. Furthermore, after optimizing the reaction conditions, we exploited the synthetic ability of TM1225 to produce ß-1,4-mannan in vitro. At 60 °C and from d-mannose 1-phosphate and mannohexaose, the enzyme synthesized mannoside chains with a degree of polymerization up to 16, which precipitated into lamellar single crystals. The X-ray powder diffraction and base-plane electron diffraction patterns of the lamellar crystals unambiguously show that the synthesized product belongs to the mannan I family previously observed in planta in pure linear mannans, such as those of the ivory nut. The in vitro formation of these mannan I crystals is likely determined by the high reaction temperature and the narrow chain length distribution of the insoluble chains.

Essential role of amino acid position 226 in oligosaccharide elongation by amylosucrase from Neisseria polysaccharea.

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosylase that synthesizes an insoluble amylose-like polymer from sole substrate sucrose. One particular amino acid, Arg226, was proposed from molecular modeling studies to play an important role in the formation of the active site topology and in the accessibility of ligands to the catalytic site. The systematic mutation of this Arg residue by all 19 other possible amino acids revealed that all single-mutants had a higher activity on sucrose compared to the wild-type enzyme. An extensive kinetic investigation showed that catalytic efficiencies are greatly impacted by the presence of natural acceptors in the reaction media, their chain length and the nature of the amino acid at position 226. Compared to the wild-type enzyme, the R226N mutant showed a 10-fold enhancement in the catalytic efficiency and a nearly twofold higher production of an insoluble amylose-like polymer that can be of interest for biotechnological applications.

Structural investigation of the thermostability and product specificity of amylosucrase from the bacterium Deinococcus geothermalis.

Amylosucrases are sucrose-utilizing α-transglucosidases that naturally catalyze the synthesis of α-glucans, linked exclusively through α1,4-linkages. Side products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes. Here, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis (DgAS). The three-dimensional structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying the DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea in complex with turanose. In the amylosucrase from N. polysaccharea (NpAS), key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanoid tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS. Our findings help to understand the inter-relationships between amylosucrase structure, flexibility, function, and stability and provide new insight for amylosucrase design.

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.

The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries.

The in vitro MutaGen procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta and two with pol eta). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol beta were 4-7-fold less mutated than those created with pol eta, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol beta and pol eta provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol eta displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomisation.

Discovery of carbamate degrading enzymes by functional metagenomics.

Bioremediation of pollutants is a major concern worldwide, leading to the research of new processes to break down and recycle xenobiotics and environment contaminating polymers. Among them, carbamates have a very broad spectrum of uses, such as toxinogenic pesticides or elastomers. In this study, we mined the bovine rumen microbiome for carbamate degrading enzymes. We isolated 26 hit clones exhibiting esterase activity, and were able to degrade at least one of the targeted polyurethane and pesticide carbamate compounds. The most active clone was deeply characterized. In addition to Impranil, this clone was active on Tween 20, pNP-acetate, butyrate and palmitate, and on the insecticide fenobucarb. Sequencing and sub-cloning of the best target revealed a novel carboxyl-ester hydrolase belonging to the lipolytic family IV, named CE_Ubrb. This study highlights the potential of highly diverse microbiota such as the ruminal one for the discovery of promiscuous enzymes, whose versatility could be exploited for industrial uses.

Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

Characterisation of a novel amylosucrase from Deinococcus radiodurans.

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.