Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

| openaire: EC/H2020/648925/EU//BHIVEAcetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono-or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.Peer reviewe

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Chapter 12: Progress toward synthetic glyconjugate vaccine against Burkholderia cepacia

International audienc

Chapter 12: Progress toward synthetic glyconjugate vaccine against Burkholderia cepacia

International audienc

Formaldehyde as a Promising C1 Source: The Instrumental Role of Biocatalysis for Stereocontrolled Reactions

In the context of the depletion of fossil resources, formaldehyde is an emerging C-1 source exhibiting high and versatile reactivity, in comparison to the most studied C-1 molecules (CO2, CO, HCOOH, and CH4). In the present mini-review, we show that biocatalysis is an ideal approach to control the reactivity of formaldehyde and to use its great potential as a platform for the synthesis of value-added chiral products. The ability of aldolases and ThDP-dependent enzymes to catalyze stereocontrolled carbon-carbon bond formation with formaldehyde are shown to involve aldol and umpolung reactivity. The synergetic combination of (i) enzyme discovery, (ii) mechanistic understanding, and (iii) enzyme engineering provides highly stereospecific biocatalysts of increasing interest for synthetic chemistry

Developping a high-throughput screening method for new tools for lignocellulosic biomass conversion

Secondary plant cell walls are the repositories of renewable lignocellulosic biomass, which is composed of cellulose, hemicelluloses (HC) and lignins. Xylans are the major HC of cell walls of many plants, including grasses and broadleaved trees, representing up to 90% of the HC in cereals and 50% in soft wood[1]. Consequently xylans, which are composed of a main-chain built of D-xylosyl units that can be substituted by avariety of chemical moieties including acetyl groups, α-D-uronic acids and/or α-L-arabinose[1], represent a major source of pentoses in biorefineries. Therefore, the optimal valorization of these is vital for the sustainability of biorefinery processes. In this context, the development of new pentose-based products and the tools and processes to manufacture them is of considerable strategic value. Xylans can be hydrolyzed by endo-β-1,4-xylanases. These enzymes cleave the glycosidic bond linking two D-xylosyl units in presence of awater molecule acting as an acceptor. However, in the presence of acceptors other than water, certain xylanases display the ability to catalyze transglycosylation reactions, leading to the synthesis of xylose-based products[2], such as xylooligosaccharides or alkylpolypentosides, both of which are potentially valuable molecules that have commercial value. Nevertheless, the yields of enzyme-driven transglycosylation reactions are often low, due to primary competition with hydrolysis and secondary hydrolysis of the synthetic product

Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 β-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria

In pressThe β-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues Asp393, Glu416 and Glu364 was performed. NMR analysis of the xyloside hydrolysis products, the change of the reaction rate-limiting step for the Glu416 mutants, the pH dependency of E416A activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining mechanism of glycoside hydrolysis, Glu416 playing the role of acid/base catalyst and Asp393 that of nucleophile

Practical synthesis of valuable d-rhamnoside building blocks for oligosaccharide synthesis

International audienceThe efficient synthesis of d-rhamnoside and the corresponding methods for its regioselective protections and deprotections have been developed in order to provide key building blocks for complex oligosaccharide syntheses toward vaccines against bacterial infections

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

International audienceCarbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In Nature, glycosidic linkages are mainly synthesized by Leloir glycosyltransferases, but can result from the action of non-Leloir transglycosylases or phosphorylases. Advantageously for chemists, non-Leloir transglycosylases are glycoside hydrolases, enzymes that are readily available and exhibit a wide range of substrate specificities. Nevertheless, non-Leloir transglycosylases are unusual glycoside hydrolases in as much that they efficiently catalyse the formation of glycosidic bonds, whereas most glycoside hydrolases favour the mechanistically related hydrolysis reaction. Unfortunately, because non-Leloir transglycosylases are almost indistinguishable from their hydrolytic counterparts, it is unclear how these enzymes overcome the ubiquity of water, thus avoiding the hydrolytic reaction. Without this knowledge, it is impossible to rationally design non-Leloir transglycosylases using the vast diversity of glycoside hydrolases as protein templates. In this critical review, a careful analysis of literature data describing non-Leloir transglycosylases and their relationship to glycoside hydrolase counterparts is used to clarify the state of the art knowledge and to establish a new rational basis for the engineering of glycoside hydrolases