Exploring the tandem placement of carbohydrate-binding domains on the enzyme activity of Photobacterium damselae sialyltransferase and Pasteurella multocida sialyltransferase.

Abstract

Carbohydrate-binding modules (CBMs) are non-catalytic domains prevalent in carbohydrate-active enzymes, including glycoside hydrolases, polysaccharide lyases, and glycosyltransferases. CBMs are well known to enhance the enzyme activity of glycoside hydrolases towards insoluble polysaccharides by proximity, targeting, and disruptive effects, and towards small, soluble oligosaccharides, likely via internal diffusion (substrate channeling). However, their effect has been relatively less studied in the context of glycosyltransferases. Here, we explored the prevalence of CBMs in sialyltransferases and assessed whether the sialyltransferase activity of the bacterial sialyltransferases Photobacterium damselae sialyltransferase (PdST) and Pasteurella multocida sialyltransferase (PmST) could be improved by tandem placement of non-native carbohydrate-binding domains that recognize the acceptor substrate. We employed three carbohydrate-binding domains (MG1D1, MN3α, and MU1α) of human gut commensal origin for this purpose and performed sialyltransferase assays using sialyltransferases fused to these domains, with lactose, lacto-N-neotetraose, and asialofetuin as acceptor substrates. The tandem presence of these non-native carbohydrate-binding domains did not significantly alter the sialylation of lactose or lacto-N-neotetraose by PdST or PmST, nor did it affect the sialylation of asialofetuin by PmST. However, the tandem placement of MN3α to PdST resulted in enhanced sialyltransferase activity towards asialofetuin. CBM-assisted engineering thus has a variable effect on the activity of sialyltransferases.

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