Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase

摘要: Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of (HA) through sequential addition single monosaccharides to non-reducing end chain. Research focused on relation between length HA oligosaccharide single-step kinetics from HA4 up HA9. It was found that turnover number kcat increased with maximum values 11 14 s-1 for NAc- UA-transfer, respectively. Interestingly, specificity constant kcat/KM polymer HA5 HA7 value 44 mM-1s-1, indicating an binding site increasing towards heptasaccharide at UA The remained moderately around 8 mM-1s-1 HA4, HA6, HA8, significantly lower NAc domain than These findings are further corroborated by structural homology model PmHAS, revealing two distinct sites oligosaccharides different sizes, one in each transferase Structural alignment studies glycosyltransferases GT-A fold showed significant similarity UDP-sugars orientation acceptor substrate. similarities substrate active essential amino acid residues involved were utilized localize sites.