Probing the Mechanism of Bacillus 1,3-1,4-β-d-Glucan 4-Glucanohydrolases by Chemical Rescue of Inactive Mutants at Catalytically Essential Residues

Abstract

The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles. While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst. Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants. Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate. The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation. E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue. Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3. By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile. Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide. Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct. This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

“…It is possible that the strong resemblance between formate and the missing carboxylate nucleophile allows better accommodation of formate in the created cavity. Similar chemical rescue of activity in the presence of azide and formate was observed for many other retaining glycoside hydrolases (20,(27)(28)(29)(30).…”

“…Interestingly, formate had a greater effect on rate acceleration, although it exhibits lower nucleophilicity. This was also observed with the nucleophile-less mutants of the ␤-glucosidase from Agrobacterium faecalis (27) and the 1,3-1,4-␤-glucanase from Geobacillus licheniformis (28). It is possible that the strong resemblance between formate and the missing carboxylate nucleophile allows better accommodation of formate in the created cavity.…”

Alteration of the Co-substrate Selectivity of Deacetoxycephalosporin C Synthase

“…The chemical rescue of mutant enzymes has been extensively investigated, although studies have focused on restoring the role of a mutated amino acid residue, e.g. by using organic amines (42,43), ions (44,45), or a modified residue involved in metal binding (46). The rescue of activity using modified (co)-substrates has been relatively underexplored, although ATP derivatives have been elegantly used to investigate the function of kinases (47).…”

Systematic identification in silico of covalently bound cell wall proteins and analysis of protein-polysaccharide linkages of the human pathogen Candida glabrata

“…Using the conserved cysteine pattern as the primary selection criterion, we identified five putative Pir proteins in C. glabrata. These five proteins also have sequence homology with known Pir proteins; therefore, we tentatively named these proteins CgPir1-5p (Hoj et al, 1992;Viladot et al, 1998). Conserved amino acid residues are given in boldface and amino acids that are identical in all sequences are boxed.…”