Is the acid/base catalytic residue mutation in β-d-mannosidase DtMan from Dictyoglomus thermophilum sufficient enough to provide thioglycoligase activity?

Glycoside hydrolases can be turned into thioglycoligase by mutation of the acid/base catalytic carboxylate residue. These mutants have proven valuable to generate S-glycosides, however, few examples in literature have described efficient thioglycoligase activity, and even fewer the underlying molecular mechanism. DtMan, a GH2 family β-d-mannosidase from the thermophilic Dictyoglomus thermophilum was cloned and expressed in E. coli. The recombinant protein is highly specific for β-d-mannosides, and exhibits efficient catalysis constants coupled to thermostability. However, seven variants bearing mutated acid/base residue could not be turned into efficient thioligases. Crystal structure of DtMan Glu425Cys mutant and molecular modeling calculations have demonstrated that unlike other GH2 thioligase reported, active site accessibility of thiol acceptor may be impaired by entrance loop rigidity. This structural feature may explain why DtMan mutants do not exhibit thioglycoligase activity. [Display omitted] •Man is a thermostable, highly specific GH2 β-d-mannosidase from Dictyoglomus thermophilum.•7 Acid/base mutants Glu425X were generated that proved to be poor yet active hydrolases without thioglycoligase activity.•Crystal structure of Glu425Cys mutant was solved and exhibited an intramolecular disulfide bridge.•Comparisons of 3D models of DtMan and of thioligase CfMan2A showed that DtMan active site was more rigid.