Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to h ave an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha -linked galactose from a pk epitope is substituted at the O6 position (Wakarchuk, W, W,, Gilbert, M,, Martin, A., Wu, Y,, Brisson, J, R,, Thibau lt, P,, and Richards, J, C, (1998) Eur. J, Biochem. 254, 626-633), Using a synthetic pk-epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E( L1)-derived sialyltransferase can make both alpha -2,3 and alpha -2,6 linka ges to the terminal galactose, Gene disruption experiments showed that the Ist gene in 126E(L1) was responsible for the in vivo addition of the alpha -2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional e nzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) alpha -2,3/6-sialyltransferase was replaced by allelic exchang e with the monofunctional MC58(L3) alpha -2,3-sialyltransferase and with th e mutant MC58(L3) allele G168I, We observed that the level of LOS sialylati on with the G168I allele was very similar to that of the wild type 126E(Ll) , indicating that residue 168 is the critical residue for the alpha -2,6-si alyltransferase activity in vitro as well as in vivo.