David L. Zechel

Dissection of nucleophilic and acid–base catalysis in glycosidases

A startling array of added anions have been observed to function as replacement catalytic nucleophiles in mutant glycosidases, including formate, azide, fluoride and other halides. Likewise, the mechanism of acid-base catalysis is somewhat plastic. The carboxylic acids can be substituted by a sulfenic acid or by ascorbate, and the effective acid strength enhanced by the introduction of strong hydrogen bonds. These studies provide an interesting bridge between enzymes and models thereof.

The E358S mutant of Agrobacterium sp. β‐glucosidase is a greatly improved glycosynthase

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The ‘original’ glycosynthase, derived from Agrobacterium sp. β‐glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583–5584]. This mutant has now been re‐cloned with a His6‐tag into a pET‐29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24‐fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP‐GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP‐β‐LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the α‐glycosyl fluoride.

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.

Understanding How Diverse β-Mannanases Recognize Heterogeneous Substrates

The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polar interactions with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.

Anatomy of Glycosynthesis: Structure and Kinetics of the Humicola Insolens Cel7B E197A and E197S Glycosynthase Mutants

The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. Glycosynthases, specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).