Essential role of glutamate 317 in galactosyl transfer by α3GalT: a computational study

Retaining glycosyltransferases (ret-GTs) are the enzymes responsible for the biosynthesis of highly specific glycosidic bonds and have drawn the interest of the scientific community. The catalytic mechanism of such enzymes is not yet fully understood and its study remains a challenge for both experimental and theoretical researches. In the case of ret-GTs where a well defined nucleophilic agent is identified in the vicinity of the anomeric center, a double-displacement mechanism via a covalent enzyme-glycosyl intermediate is commonly assumed and has received some experimental support, although not direct and univocal evidence has been obtained so far. This is the case for α-(1→3)-galactosyltransferase (α3GalT), a ret-GT from Bos taurus where a glutamate (Glu317) is in suitable position to act as a nucleophile. Here we perform density functional theory (DFT) quantum mechanics/molecular mechanics (QM/MM) calculations on the full α3GalT enzyme to analyze the role of Glu317 in the catalytic process. This is done not only for the double-displacement mechanism, where the function of the nucleophile is obvious, but also in the scenario of a front-side attack mechanism (via an oxocarbenium ion-like transition state (S(N)i) or an ion-pair oxocarbenium intermediate (S(N)i-like)). Glu317 is found to be essential in both cases. For a front-side attack, this residue would have a key role in leaving group departure and consequent stabilization of the increasing positive charge at the anomeric center. This finding alerts on the interpretation of the mutagenesis data as both, the formation of a covalent intermediate and a S(N)i or a S(N)i-like mechanism 'assisted' by a nucleophile, could be consistent with experiment. In addition, it could explain why the covalent enzyme-glycosyl intermediate has never been isolated.