A new multifunctional α2,3-sialyltransferase has been discovered in Pasteurella dagmatis. The enzyme, in short PdST, was identified from the P. dagmatis genome by sequence similarity with sialyltransferases of glycosyltransferase family GT-80. In addition to its regioselective sialyltransferase activity (5.9 U/mg; pH 8.0), purified PdST is alternatively active at low pH as α2,3-sialidase (0.5 U/mg; pH 4.5) and α2,3-trans-sialidase (1.0 U/mg; pH 4.5). It also shows cytidine-5'-monophosphate N-acetyl-neuraminic (CMP-Neu5Ac) hydrolase activity (3.7 U/mg; pH 8.0) when no sialyl acceptor substrate is present in the reaction. After sialyltransferase PmST1 from P. multocida, PdST is the second member of family GT-80 to display this remarkable catalytic promiscuity. A unique feature of PdST, however, is a naturally occurring Ser-to-Thr substitution within a highly conserved Y(112)DDGS(116) sequence motif. In PmST1, the equivalent Ser(143) is involved in binding of the CMP-Neu5Ac donor substrate. Reversion of the natural mutation in a T116S-PdST variant resulted in a marked increase in α2,3-trans-sialidase side activity (4.0 U/mg; pH 4.5), whereas the major sialyltransferase activity was lowered (3.8 U/mg; pH 8.0). The Michaelis-Menten constant for CMP-Neu5Ac was decreased 4-fold in T116S mutant when compared with wild-type PdST (KM=1.1 mM), indicating that residue 116 of PdST contributes to a delicate balance between substrate binding and catalytic activity. D-Galactose and various β-D-galactosides function as sialyl acceptors from CMP-Neu5Ac, whereas other hexoses (e.g. D-glucose) are inactive. Structure comparison was used to rationalize the particular acceptor substrate specificity of PdST in relation to other GT-80 sialyltransferases that show strict α2,3-regioselectivity, but are flexible in using α/β-galactosides for sialylation.
Keywords: N-terminal elongation; bacterial sialyltransferase; glycosyltransferase family GT-80; natural mutation; structure–function relationships.