To date, very little is known about anabolic glycosyl transfer reactions involved in the formation of polysaccharides. However, it has been predicted that this type of reaction would mechanistically be the reverse of glycosyl hydrolysis (28
). Based on the extensively characterized mechanism of glycosyl hydrolase, a mechanism for glycosyl transfer has been proposed (26
). The mechanism of the β-glycosyl transfer reaction involves a single nucleophilic substitution at the anomeric center. This catalytic mechanism involves a single acidic active-site amino acid that acts as a nucleophile (26
). Previous studies of glycosyltransferases have demonstrated that there is often insufficient sequence similarity for functional predictions with traditional sequence alignments. However, it is suggested that certain conserved regions may be required for a common function between the various transferases (26
). HCA is a powerful sequence comparison method which can clearly detect three-dimensional similarities in proteins showing very limited sequence relatedness (6
). This method also has enabled the prediction of catalytic residues in a number of glycosyl hydrolases by the identification of invariant amino acid residues with appropriate side chain reactivity (10
). Using HCA, Saxena et al. (26
) compared 13 β-glycosyltransferases and identified a conserved domain A that should be directly involved in the formation of a single β-glycosidic linkage from α-linked nucleotide diphospho-sugar donors. They characterized the Asp residue at the C-terminal end of this domain as a catalytic residue. Keenleyside and Whitfield identified 32 proteins, in addition to the 13 originally described by Saxena et al., that possess this conserved domain (18
On the other hand, α-glycosyltransferases retain the anomeric configuration at the reaction center via a two-step mechanism (26
). This mechanism involves two acidic active-site amino acids, one acting as a nucleophile and the other acting as a general base. The first step of the nonprocessive α-glycosyl transfer reaction involves the attack of an enzymatic nucleophile on the anomeric center of the sugar, leading to formation of a β-glycosyl enzyme intermediate. The distance between the catalytic nucleophile and the anomeric carbon of UDP-hexose is smaller than that in β-glycosyltransferases, where a larger distance is required to accommodate the acceptor molecule between the catalytic base and the anomeric carbon. However, the catalytic mechanism of the first step of α-glycosyl transfer reaction includes the formation of β-glycosidic linkage, and this action is similar to that of β-glycosyltransferase. Thus, it is to be expected that nonprocessive α-glycosyltransferases possess the domain previously identified in the β-glycosyltransferases by Saxena et al. (26
). The conserved domain of WaaO and other homologous proteins was similar to that of β-glycosyltransferases. The DXD motif in region I was located at the C-terminal end of the domain. This DXD motif was also conserved in almost all β-glycosyltransferases described by Saxena et al. (26
) and Keenleyside and Whitfield (18
). This motif includes a conserved Asp residue previously characterized as a possible catalytic residue by Saxena et al. (26
). This motif falls in a loop at the C-terminal ends of predicted strands, a position frequently observed for catalytic residues (31
). Taken together, the conserved domain and region I are likely to be important for the formation of β-glycosidic linkage between donor sugar and the enzyme.
Regions II, III, and IV are present in only WaaO and the homologous glycosyltransferases. Given their common activities and the conservation of these regions, it is likely that these regions represent at least part of the catalytic or binding sites and play a crucial role in generating α-glycosidic linkage between donor sugar and acceptor molecule.
The results of site-directed mutagenesis analysis of WaaO indicate that Asp-131 and -133 (DXD motif in region I) and Asp-220 and -222 (DXD motif in region III) may play a crucial role in the catalytic function of E. coli K-12 WaaO protein.
The present analysis suggests that the nonprocessive α-glycosyltransferases that were examined constitute a single protein family. These proteins have four conserved regions and a single domain, each presumably involved in the formation of α-glycosidic linkage between donor sugar and acceptor molecule. The lack of this conserved architecture in other α-glycosyltransferases indicates the presence of more than a single family in this class of enzymes, as previously described by Campbell et al. (3