Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

Abstract

Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'‐diphosphoglucose (UDP‐Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X‐ray diffraction analysis of the PaGS/UDP‐Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose‐like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose‐like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5‐anhydro‐D‐arabino‐hex‐1‐enitol (AA) or its tautomeric form, 1,5‐anhydro‐D‐fructose. These findings are consistent with a stepwise SNi‐like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism. © 2012 IUBMB Life, 64(7): 649–658, 2012.

The Crystal Structure of Nitrosomonas europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into Sucrose Metabolism in Prokaryotes

“…S10 in the supplemental material). This ligand mimics the planar structure of the transition state in either the S N i, S N i-like, or elimination/addition mechanism (56). This type of in situ-generated intermediate was also observed in the E. coli and P. abyssi glycogen synthases (25,56).…”

“…In either of those two cases, an important stabilization of the transition state would be based on the interaction between the anomeric carbon (C-1) of the sugar being transferred and the oxygen of the main chain of a His residue (24,55). Recently, an alternative elimination/addition mechanism has been proposed for the Pyrococcus abyssi glycogen synthase (56), which was argued to be compatible with the currently available data. In this mechanism, a general base is needed to extract a proton from C-2 of the glycosyl group.…”

“…This ligand mimics the planar structure of the transition state in either the S N i, S N i-like, or elimination/addition mechanism (56). This type of in situ-generated intermediate was also observed in the E. coli and P. abyssi glycogen synthases (25,56). Regardless of these alternative mechanisms, it must be critical that the main-chain oxygen of H425 in N. europaea sucrose synthase is near C-1/C-2 in the transition state.…”

“…This mechanism in which the catalytic residues get into places upon closing may explain why it is not trivial to obtain a closed structure with an intact sugar nucleotide. For instance, crystal structures of the closed forms were obtained for the E. coli glycogen synthase and the A. thaliana sucrose synthase grown in presence the sugar nucleotide, but the glycosyl group was slowly cleaved (23,25,56). There are other retaining GT-B structures with a sugar nucleotide bound, but those were described as "semiclosed" (44).…”

“…However, not all of them may require such a large conformational change. The glycogen synthase was another case with closed and wide open structures described (25,47,56). The sucrose-6-phosphate synthase must also have the same type of behavior, but only an open structure is available (61).…”