Pasturella multocida heparosan synthase 2 (PmHS2) is a widely used biocatalyst for synthesizing heparosan and heparan sulfate derivatives. It is a bi-functional glycosyltransferase containing α1-4-N-acetylglucosaminyltransferase and β1-4-glucuronyltransferase. Heparan sulfate is a complex, highly anionic, linear polysaccharide and plays an essential biological role as a multicellular regulator. Heparan sulfate regulates a wide array of cellular activities through binding with more than 100 heparan sulfate-binding proteins. Yet, a detailed study regarding how the structural motif of heparan sulfate leads to versatile biological activities has not been fully elucidated. It thus highlights the need for engineering PmHS2 to achieve structurally defined heparan sulfate in large quantities, furthermore to understand the structure-activity relationship of heparan sulfate. Here, we report recent efforts to engineer PmHS2, which leads to synthesize heparosan decasaccharide in a size-controlled manner along with to expand substrate promiscuity for achieving vast chemical space of heparan sulfate. The removal of an N-terminal 80 amino acid sequence improved not only its thermal stability but also its expression level by 3-4 fold. Taking advantage of improved properties, Δ80PmHS2 led to the gram scale of synthesis of heparosan hexasaccharide. We also identified for the first time the reverse glycosylation activities of PmHS2, which is a first report on polysaccharide synthase. The reverse glycosylation activity of PmHS2 limited its application in size-controlled synthesis of longer oligosaccharide. The key catalytic residues of both domains were successfully identified through structure alignment. The generating mono-functional PmHS2 through mutating key residues minimized side product formation from reverse glycosylation, leading to synthesis on decasaccharide in quantitative yield. Currently, the possible amino acid residues of PmHS2 associated with substrate recognition have been identified through structural alignment. Mutagenesis on the identified residues is in progress for expanding substrate promiscuity.

Engineering polysaccharide synthases toward size-controlled synthesis of heparosan oligosaccharide and expanding chemical space of heparan sulfate analogs

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.