GD3-synthase is a sialyltransferase that catalyzes the synthesis of ganglioside GD3 leading to the b- and c-series gangliosides. It contains four common sequence regions of vertebrate sialyltransferases, referred to as the L, S, 111, and VS sialylmotifs, which have been identified in all vertebrate sialyltransferases that play important roles in spatial structure maintenance and protein functions. No 3D structural information, however, is currently available for vertebrate sialyltransferases. Using primary sequence of human GD3-synthase, we identified the structure of a prokaryotic sialyltransferase (Cstll, also known as an alpha 2,3/ a2,8 -sialyltransferase) as the template for protein homology modeling. Secondary structural alignment between these two proteins identified several conserved amino-acid residues. The functions of four conserved residues (Asn(188), Pro(189), Set(190), and Arg(272)) between the L and S sialylmotifs in human GD3-synthase were investigated using mutational analysis and molecular modeling, and it was found that these sites are involved in determining the (alpha 2,8-linkage specificity of GD3-synthase. (c) 2008 Elsevier Inc. All rights reserved.