Dominik Stoll

β-Mannosynthase: Synthesis of β-Mannosides with a Mutant β- Mannosidase

Engineering enzymes: The glutamic acid nucleophile of a retaining β-mannosidase has been replaced with a serine residue to form a β-mannosynthase. When the new enzyme is provided with an α-mannosyl fluoride donor and an appropriate acceptor, β-mannoside linkages are synthesized. Remarkably, α-mannosyl fluoride can be generated in situ by providing the mannosynthase with excess fluoride ion.

Expansion of the glycosynthase repertoire to produce defined manno-oligosaccharides

Mutant endo-mannanases, in which the catalytic nucleophile has been replaced, function as glycosynthases in the synthesis of manno-oligosaccharides of defined lengths.

Characterization of a β‐N‐acetylhexosaminidase and a β‐N‐acetylglucosaminidase/β‐glucosidase from Cellulomonas fimi

The Gram‐positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular β‐N‐acetylglucosaminidases, a family 20 β‐N‐acetylhexosaminidase (Hex20), and a novel family 3‐β‐N‐acetylglucosaminidase/β‐glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 °C were 0.066 s−1·mm−1 and 0.076 s−1·mm−1 for 4′‐nitrophenyl β‐N‐acetyl‐d‐glucosaminide and 4′‐nitrophenyl β‐d‐glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between β‐N‐acetylglucosaminidases and β‐glucosidases of family 3. Reaction by a double‐displacement mechanism was confirmed for Nag3 through the identification of a glycosyl–enzyme species trapped with the slow substrate 2′,4′‐dinitrophenyl 2‐deoxy‐2‐fluoro‐β‐d‐glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave β‐glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the d‐glucosyl/d‐galactosyl configuration of the glycone: Km and kcat values were 53 µm and 482.3 s−1 for 4′‐nitrophenyl β‐N‐acetyl‐d‐glucosaminide and 66 µm and 129.1 s−1 for 4′‐nitrophenyl β‐N‐acetyl‐d‐galactosaminide.

Identification of Glu-519 as the catalytic nucleophile in β-mannosidase 2A from Cellulomonas fimi

Incubation of the beta-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta-D-mannosyl fluoride (2FMan beta F) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(i)=0.57 min(-1), dissociation constant for the inactivator K(i)=0.41 mM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-mannosyl-beta-mannosidase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glc beta(1-6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan-Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan-Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519-Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a beta-mannosidase.

Kinetics and stereochemistry of the Cellulomonas fimi β-mannanase studied using 1H-NMR

Endo–1,4-β-mannanases (β-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The β-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 β-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by 1H-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10−5s−1 at 5°C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many ‘retaining’ enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the −3 subsite had a greater impact on the apparent kcat/Km ratio (the catalytic efficiency) than the +2 subsite. The β-anomer of mannotriose was hydrolysed 1000-times more efficiently than the α-anomer indicating selectivity for the β- over the α-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.