Abstract
Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligosaccharide and the single-step elongation kinetics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the specificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccharide at the UA domain. The value of kcat/KM remained moderately constant around 8 mM-1s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These findings are further corroborated by a structural homology model of PmHAS, revealing two distinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the acceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate binding were utilized to localize the two HA oligosaccharide binding sites.
Highlights
Enzymatic production of glycosaminoglycans has increasingly attracted attention over the last two decades as these polysaccharides are applied multifold in pharmacy and cosmetics
It was demonstrated that, for Streptococcus zooepidemicus, overexpression of genes involved in UDP-GlcNAc biosynthesis increased the molecular weight of the HA products [22], indicating that the chain length is controlled by the availability of the substrates
A combination of kinetic characterization and structural modeling was employed in order to study the polymerization of hyaluronan by Pasteurella multocida hyaluronan synthase (PmHAS), focusing on the influence of the oligosaccharide length on the turnover number and the specificity constant
Summary
Enzymatic production of glycosaminoglycans has increasingly attracted attention over the last two decades as these polysaccharides are applied multifold in pharmacy and cosmetics. It was demonstrated that, for Streptococcus zooepidemicus, overexpression of genes involved in UDP-GlcNAc biosynthesis increased the molecular weight of the HA products [22], indicating that the chain length is controlled by the availability of the substrates This is corroborated by kinetic data from HAS enzymes of different sources, demonstrating a significantly larger KMNAc value than KMUA value [23,24,25,26]. The addition of HA oligosaccharides to the reaction in the presence of both UDP-sugars exacerbates the polymerization rate of PmHAS and diminishes the polydispersity of the HA products compared to reactions initiated with only the UDP-sugars [28] These findings have resulted in increased molecular weight products with minimal polydispersity, the kinetic elongation mechanism of HAS enzymes supporting these results remains unknown. This study presents evidence for two distinct oligosaccharides binding sites within PmHAS which affects the polydispersity of the HA products
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