Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase
Author(s)
Floor K. Kooy,
Hendrik H. Beeftink*,
Michel H. M. Eppink*,
Johannes Tramper,
Gerrit Eggink,
Carmen G. Boeriu
Affiliation(s)
Food and Biobased Research, Wageningen University and Research Center, Wageningen, The Netherlands. Bioprocess Engineering, Wageningen University and Research Center, Wageningen, The Netherlands.
Food and Biobased Research, Wageningen University and Research Center, Wageningen, The Netherlands. Bioprocess Engineering, Wageningen University and Research Center, Wageningen, The Netherlands.
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.
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.
Cite this paper
Kooy, F. , Beeftink, H. , Eppink, M. , Tramper, J. , Eggink, G. and Boeriu, C. (2013) Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase. Advances in Enzyme Research, 1, 97-111. doi: 10.4236/aer.2013.14011.
Kooy, F. , Beeftink, H. , Eppink, M. , Tramper, J. , Eggink, G. and Boeriu, C. (2013) Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase. Advances in Enzyme Research, 1, 97-111. doi: 10.4236/aer.2013.14011.
References
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(1997) Culture conditions affect the molecular weight properties of hyaluronic acid produced by Streptococcus zooepidemicus. Applied & Environmental Microbiology, 63, 2759-2764. [13] Widner, B., et al. (2005) Hyaluronic acid production in Bacillus subtilis. Applied & Environmental Microbiology, 71, 3747-3752. http://dx.doi.org/10.1128/AEM.71.7.3747-3752.2005 [14] DeAngelis, P.L., Papaconstantinou, J. and Weigel, P.H. (1993) Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria. Journal of Biological Chemistry, 268, 14568-14571. [15] Chien, L.-J. and Lee, C.-K. (2007) Hyaluronic acid production by recombinant Lactococcus lactis. Applied Microbiology and Biotechnology, 77, 339-346. http://dx.doi.org/10.1007/s00253-007-1153-z [16] Yu, H. and Stephanopoulos, G. (2008) Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid. 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