In Vitro Synthesis and Crystallization of β-1,4-Mannan

In vitro polymerization of β-mannans is a challenging reaction due to the steric hindrance confered by the configuration of mannosyl residues and the thermodynamic instability of the β-anomer. Whatever the approach used to date—whether chemical, or enzymatic with glycosynthases and mannosyltransferases—pure β-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 β-1,4-mannan. The recombinant production of this enzyme and its biochemical characterization allowed us to prove that it catalyzes the reversible phosphorolysis of β-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 β-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

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 α-1,6 and α-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 α-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

Deciphering an Undecided Enzyme: Investigations of the Structural Determinants Involved in the Linkage Specificity of Alternansucrase

Understanding how polymerases catalyze the synthesis of biopolymers is a timely and important issue in generating controlled structures with well-defined properties. With this objective in mind, here we describe the 2.8 angstrom crystal structure of a truncated version of alternansucrase (ASR) from L. citreum NRRL B-1355. Indeed, ASR is a striking example of alpha-transglucosylase among GH70 glucansucrases, capable of catalyzing high and low molar mass alternan, an alpha-glucan comprising alternating alpha-1,3 and alpha-1,6 linkages in its linear chain. The 3D structure sheds light on the various features involved in enzyme stability. Moreover, docking studies and biochemical characterizations of 17 single mutants and two double mutants enable the key determinants of alpha-1,6 or alpha-1,3 linkage specificity to be located and establish the structural basis of alternance. ASR displays two different acceptor subsites in the prolongation of its subsites -1 and +1. The first one is defined by Trp675, a residue of subsite +2, and orients acceptor binding exclusively toward alpha-1,6 linkage synthesis. The second binding site comprises Asp772 and Trp543, two residues defining the +2' and +3' subsites, respectively, which are critical for alpha-1,3 linkage formation. It is proposed that the interplay between these two acceptor sites controls alternance. These results add to the toolbox of enzymes for the production of tailor-made polysaccharides with controlled structures

Glucansucrases: Structural basis, mechanistic aspects, and new perspectives for engineering

International audienc

Extending the Structural Diversity of alpha-Flavonoid Glycosides with Engineered Glucansucrases

International audienceFlavonoids constitute an important class of bioactive molecules, the physicochemical properties of which can be modulated by glucosylation. A structurally guided approach has been used to isolate glucansucrases modified in their acceptor-binding site and specialized for luteolin glucosylation. Of a small-size library, we isolate mutants showing up to an 8-fold increase in flavonoid conversion rate over that observed with the parental enzyme. Di- and triglucosylated luteolin derivatives never described before have been obtained. They exhibit 282- and 17708-fold increases in water solubility, respectively, and are protected from oxidation by the glucosylation reaction. Molecular docking enables insight into the product specificity of the best mutants. These results demonstrate that atransglucosylase engineering is a powerful means to generate highly specific catalysts for flavonoid glucosylation and deliver new structural scaffolds with increased bioavailability and high relevance for therapeutic applications

Le Courrier

06 mars 18511851/03/06 (N65)

Structural Investigation of the Thermostability and Product Specificity of Amylosucrase from the BacteriumDeinococcus geothermalis

International audienceBackground: Amylosucrases (AS) hold great potential for glycodiversification. Results: The first three-dimensional structure of AS from Deinococcus geothermalis solved here revealed an unusual dimer organization. Structures of complex of AS with turanose were also determined. Conclusion: Dimerization may contribute to thermostability. Turanose versus trehalulose formation is controlled by residues from subsite ϩ1. Significance: This study improves the comprehension of AS properties and provides new insight for AS design

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

International audienceThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio-and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN 123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN 123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the interrelationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens. Carbohydrate-active enzymes catalyze a wide range of chemical reactions. They have emerged as a practical alternative to chemical catalysts, avoiding multiple steps of protection and deprotection often required in chemical synthesis to control the reactivity of the sugar hydroxyl groups and regio-and stereo-selectivity of the reaction. Some of them are rather versatile biocatalysts often displaying naturally a relaxed substrate specificity. This promiscuity can be further exacerbated by enzyme engineering to either broaden or narrow down the range of recognized substrates and/or control the reaction selectivity 1. In particular, mutagenesis targeting the enzym

ORFs of interest from the sequenced clones, annotated as putative esterases or proteases.

<p>ORFs of interest from the sequenced clones, annotated as putative esterases or proteases.</p