A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

Microbial ?-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) fromLeuconostoc citreumNRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASR?2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln(700)and Tyr(717)) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains

Development and characterization of multi-enzymatic system for lignocellulose degration

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An eco-design approach for an innovative production process of low molar mass dextran

An approach for early-stage eco-design of an enzyme-based process has been developed by coupling process modeling, Life Cycle Assessment (LCA) and flowsheet design, in order to evaluate the real advantages of the direct synthesis of low molar mass dextrans (5-25 kg mol(-1)) from a sucrose substrate. This approach identifies the most promising development pathways and crucial unit operations that require, as a matter of priority, further investigation and experimentation. Process modeling is based on a comprehensive and multi-fidelity building of Life Cycle Inventories (LCI) to establish all materials and energy inputs and outputs of the processes involved with a flexible and satisfactory level of accuracy. This essentially binds (i) a high-fidelity polymerization model, namely PolyEnz, for the description of the synthesis of dextrans from sucrose using the DSR-M enzyme following a non-processive mechanism, (ii) flexible-fidelity models for description of subsequent purification, separation and drying steps, (iii) upstream processes in the value chain and life cycle system using real-world data from ecoinvent datasets. Three process benchmarks were constructed and compared to determine the most appropriate purification processes and operation conditions at a larger scale. In addition, various process triggers, including the initial concentration and type of substrate, the type of process water, the use of size exclusion chromatography for separation, and the use of freeze drying for the last production stage were subjected to a sensitivity analysis with the criteria being the overall energy demand, the potential environmental damage evaluated by the ReCiPe endpoint and the global warming potential

Investigations on the Determinants Responsible for Low Molar Mass Dextran Formation by DSR-M Dextransucrase

Certain enzymes of the GH70 family dextransucrases synthesize very high molar mass dextran polymers, whereas others produce a mixed population of very high and low molar mass products directly from sucrose substrate. Identifying the determinants dictating polymer elongation would allow the tight control of dextran size. To explore this central question, we focus on the recently discovered DSR-M enzyme from Leuconostoc citreum NRRL B-1299, which is the sole enzyme that naturally, exclusively, and very efficiently produces only low molar mass dextrans from sucrose. Extensive biochemical and structural characterization of a truncated form of DSR-M (DSR-MΔ2, displaying the same biochemical behavior as the parental enzyme) and X-ray structural analysis of complexes with sucrose and isomaltotetraose molecules together with accurate monitoring of the resulting polymer formation reveal that DSR-MΔ2 adopts a nonprocessive mechanism attributed to (i) a high propensity to recognize sucrose as a preferred acceptor at the initial stage of catalysis, (ii) an ability to elongate oligodextrans irrespective of their size, and (iii) the presence of a domain V showing a weak ability to bind to the growing dextran chains. In this study, we present the 3D structure with the largest defined domain V reported to date in the GH70 family and map sugar binding pockets on the basis of the structure of the complex obtained with isomaltotetraose. Altogether, these findings give insights into the interplay between the domain V and the catalytic site during polymerization. They open promising strategies for GH70 enzyme engineering aiming at modulating glucan size

Branching pattern of gluco-oligosaccharides and 1.5 kDa dextran grafted by the alpha-1,2 branching sucrase GBD-CD2

GBD-CD2, an engineered sucrose-acting enzyme of glycoside hydrolase family 70, transfers D-glucopyranosyl (D-Glcp) units from sucrose onto dextrans or gluco-oligosaccharides (GOS) through the formation of alpha-(1 -> 2) linkages leading to branched products of interest for health, food and cosmetic applications. Structural characterization of the branched products obtained from sucrose and pure GOS of degree of polymerization (DP) 4 or DP 5 revealed that highly alpha-(1 -> 2) branched and new molecular structures can be synthesized by GBD-CD2. The formation of alpha-(1 -> 2) branching is kinetically controlled and can occur onto vicinal alpha-(1 -> 6)-linked D-Glcp residues. To investigate the mode of branching of 1.5 kDa dextran, simulations of various branching scenarios and resistance to glucoamylase degradation were performed. Analysis of the simulation results suggests that the branching process is stochastic and indicates that the enzyme acceptor site can accommodate both linear and poly-branched acceptors. This opens the way to the design of novel enzyme-based processes yielding carbohydrate structures varying in size and resistance to hydrolytic enzymes. (C) 2013 Elsevier Ltd. All rights reserved

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

Investigating the function of an arabinan utilization locus isolated from a termite gut community

Biocatalysts are essential for the development of bioprocesses efficient for plant biomass degradation. Previously, a metagenomic clone containing DNA from termite gut microbiota was pinpointed in a functional screening that revealed the presence of arabinofuranosidase activity. Subsequent genetic and bioinformatic analysis revealed that the DNA fragment belonged to a member of the genus Bacteroides and encoded 19 open reading frames (ORFs), and annotation suggested the presence of hypothetical transporter and regulator proteins and others involved in the catabolism of pentose sugar. In this respect and considering the phenotype of the metagenomic clone, it was noted that among the ORFs, there are four putative arabinose-specific glycoside hydrolases, two from family GH43 and two from GH51. In this study, a thorough bioinformatics analysis of the metagenomic clone gene cluster has been performed and the four aforementioned glycoside hydrolases have been characterized. Together, the results provide evidence that the gene cluster is a polysaccharide utilization locus dedicated to the breakdown of the arabinan component in pectin and related substrates. Characterization of the two GH43 and the two GH51 glycoside hydrolases has revealed that each of these enzymes displays specific catalytic capabilities and that when these are combined the enzymes act synergistically, increasing the efficiency of arabinan degradation

A convergent chemoenzymatic strategy to deliver a diversity of Shigella flexneri serotype-specific O-antigen segments from a unique lightly protected tetrasaccharide core

International audienceProgress in glycoscience is strongly dependent on the availability of broadly diverse tailored-made, well-defined and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrheal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization post enzymatic -D-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the Glycoside Hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof-of-concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemo-enzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective -D-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest