Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue.

Lipopolysaccharyl-α-l,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 α-l,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric con- figuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carbox- ylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the Km for UDP-galactose is not significantly altered, the kcat was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surpris- ingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 Å away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a kcat value 3000-fold lower than that of the wild type enzyme. A 2.6-Å crystal structure of the Q189E mutant with bound uridine 5′-diphospho-2-deoxy-2-fluoro- α-D-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby re- kindling a longstanding mechanistic debate. [ABSTRACT FROM AUTHOR]