Exploration of Strategies for Mechanism‐Based Inhibitor Design for Family GH99 endo‐α‐1,2‐Mannanases

Abstract

endo‐α‐1,2‐Mannosidases and ‐mannanases, members of glycoside hydrolase family 99 (GH99), cleave α‐Glc/Man‐1,3‐α‐Man‐OR structures within mammalian N‐linked glycans and fungal α‐mannan, respectively. They are proposed to act through a two‐step mechanism involving a 1,2‐anhydrosugar “epoxide” intermediate incorporating two conserved catalytic carboxylates. In the first step, one carboxylate acts as a general base to deprotonate the 2‐hydroxy group adjacent to the fissile glycosidic bond, and the other provides general acid assistance to the departure of the aglycon. We report herein the synthesis of two inhibitors designed to interact with either the general base (α‐mannosyl‐1,3‐(2‐aminodeoxymannojirimycin), Man2NH2DMJ) or the general acid (α‐mannosyl‐1,3‐mannoimidazole, ManManIm). Modest affinities were observed for an endo‐α‐1,2‐mannanase from Bacteroides thetaiotaomicron. Structural studies revealed that Man2NH2DMJ binds like other iminosugar inhibitors, which suggests that the poor inhibition shown by this compound is not a result of a failure to achieve the expected interaction with the general base, but rather the reduction in basicity of the endocyclic nitrogen caused by introduction of a vicinal, protonated amine at C2. ManManIm binds with the imidazole headgroup distorted downwards, a result of an unfavourable interaction with a conserved active site tyrosine. This study has identified important limitations associated with mechanism‐inspired inhibitor design for GH99 enzymes.

Mannosidase mechanism: at the intersection of conformation and catalysis

“…Interestingly, this transition state conformation is very close to the 2 H3/E3 conformation observed upon binding of -mannosyl-1,3-mannoimidazole (see Fig. 3) [57].…”

Distortion of mannoimidazole supports a B2,5 boat transition state for the family GH125 α-1,6-mannosidase from Clostridium perfringens

“…23 However, analysis of published complexes with GH125 enzymes shows that the general acid residue is located below the mean plane of the ring, 18,19 suggesting that there are poor prospects for binding of MIm to CpGH125. Yet, there are now several examples of glycoimidazoles binding to glycosidases with good affinity even without achieving a prototypical interaction between the enzymatic general acid and the imidazole nitrogen, such as β-glucosidases of family GH116, 24 α-mannanases of family GH99 25 and α-mannosidases of family GH47 10 (Fig. 2d).…”

“…We highlight one important anomalous case, in which a complex of a disaccharide MIm analogue with BxGH99 1,2-α-mannanase revealed an unusual 2 H 3 /E 3 conformation possibly due to steric clashes with an active site residue, Tyr252. 25 In this case no K D value could be measured by ITC indicating poor affinity for the enzyme.…”

An Epoxide Intermediate in Glycosidase Catalysis

“…However, 3-dimensional structures of wild-type BtGH99 in complex with the isofagomine-derived inhibitors GlcIFG 18 and ManIFG, 12 and a disabled noncatalytic mutant in complex with a substrate, 12 failed to identify a likely enzymatic nucleophile appropriately situated near the fissile anomeric bond. Enticingly, complexes with deoxymannojirimycin-derived inhibitors GlcDMJ 18 and Man-2-amino-DMJ, 19 noeuromycin-derived inhibitors GlcNOE 18 and ManNOE, 20 and of the E333Q mutant with substrate, 12 highlighted an interaction between a carboxylate residue and the 2-hydroxyl of the −1 mannosyl residue. Collectively, these data were suggestive of an alternative mechanism involving a 1,2-anhydro sugar intermediate (Figure 1b); however, no studies have provided direct evidence for participation of O2 or insight into the proposed epoxide intermediate.…”