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

F.K. Kooy, H.H. Beeftink, M.H.M. Eppink, J. Tramper, G. Eggink, C.G. Boeriu

Research output: Contribution to journalArticleAcademicpeer-review


Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a ß1,3-glucuronyltransferase and ß1,4-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 oligo- saccharide and the single-step elongation ki- netics 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 spe- cificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1·s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccha- ride at the UA domain. The value of kcat/KM re- mained moderately constant around 8 mM-1·s-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 find- ings are further corroborated by a structural homology model of PmHAS, revealing two dis- tinct 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 ac- ceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate bind- ing were utilized to localize the two HA oligo- saccharide binding sites.
Original languageEnglish
Pages (from-to)97-111
JournalAdvances in Enzyme Research
Issue number4
Publication statusPublished - 2013


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