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

Floor K. Kooy, Hendrik H. Beeftink, Michel H. M. Eppink, Johannes Tramper, Gerrit Eggink, Carmen G. Boeriu

Bioprocess Engineering, Wageningen University and Research Center, Wageningen, The Netherlands.
Food and Biobased Research, Wageningen University and Research Center, Wageningen, The Netherlands.

DOI: 10.4236/aer.2013.14011                

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 k_cat/K_M 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 k_cat/K_M 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.

Keywords

Pasteurella; Hyaluronan; Binding Site; Polymerization; Co-Polymers

Conflicts of Interest

The authors declare no conflicts of interest.

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