Mendoza, Fernanda; Gomez, Hansel; Lluch, Jose M.; Masgrau, Laura
alpha 1,4-N-Acetylhexosaminyltransferase EXTL2: The Missing Link for Understanding Glycosidic Bond Biosynthesis with Retention of Configuration
ACS CATALYSIS, 6:2577-2589, APR 2016

Glycosyltransferases (GTs) are biocatalysts that synthesize a wide variety of glycans present in nature. The mechanism by which they form a new glycosidic bond, preserving the configuration at the anomeric carbon, has been strongly debated. In the last few years, new experimental and computational results have provided very valuable knowledge: the proposed front-side reaction seems to be the preferred path, whereas some systems (family GT6 members) can switch mechanism toward the double displacement. However, why would retaining GTs have evolved to catalyze sugar transfer by different mechanisms? The present QM(DFT)/MM work on alpha 1,4-N-acetylhexosaminyltransferase (EXTL2) fills this gap in the understanding of retaining glycosyltransferases catalysis. We show that EXTL2, despite having a nucleophilic residue (Asp246) that is structurally analogous to the Glu nucleophile found in GT6, clearly follows a front-side mechanism. Our results show that Asp246 is used to facilitate UDP GalNAc bond cleavage by stabilizing the oxocarbenium species and to compensate for the close presence of a positively charged residue (Arg293) that interacts electrostatically with the acceptor substrate carboxylate (and with Asp246). Altogether, a more complete picture of retaining GT catalysis can now be provided: the front-side mechanism is the preferred path, with the crucial contribution from substrate-assisted catalysis in facilitating UDP departure; in some cases though, an extra stabilization of the forming oxocarbenium species is needed, which is achieved by the presence of a nucleophilic residue (nucleophilically assisted catalysis). This can eventually shift (not switch) the mechanism toward a double displacement, a strategy that within this context may be regarded as an extreme case of oxocarbenium stabilization. Importantly, we also conclude that the identity of the residues found on the beta-face of the sugar in retaining GTs is strongly linked to both the required binding orientation of the acceptor substrate (for regiospecificity) and to its chemical identity (both aspects having also an influence on UDP stabilization). These are aspects that may help in the engineering of enzymes for glycan synthesis.


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