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

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

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.

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

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.

References

[1]   Gandhi, N.S. and Mancera, R.L. (2008) The structure of glycosaminoglycans and their interactions with proteins. Chemical Biology & Drug Design, 72, 455-482. http://dx.doi.org/10.1111/j.1747-0285.2008.00741.x

[2]   Schaefer, L. and Schaefer, R.M. (2010) Proteoglycans: From structural compounds to signaling molecules. Cell and Tissue Research, 339, 237-246. http://dx.doi.org/10.1007/s00441-009-0821-y

[3]   Stern, R. (2008) Association between cancer and “acid mucopolysaccharides”: An old concept comes of age, finally. Seminars in Cancer Biology, 18, 238-243. http://dx.doi.org/10.1016/j.semcancer.2008.03.014

[4]   Meyer, K. and Palmer, J.W. (1934) The polysaccharide of the vitreous humor. Journal of Biological Chemistry, 107, 629-634.

[5]   Boas, N.F. (1949) Isolation of hyaluronic acid from the cock’s comb. Journal of Biological Chemistry, 181, 573-575.

[6]   Meyer, K. and Chaffee, E. (1941) The mucopolysaccharides of skin. Journal of Biological Chemistry, 138, 491-499.

[7]   Chain, E. and Duthie, E.S. (1940) Identity of hyaluronidase and spreading factor. British Journal of Experimental Pathology, 21, 324-338.

[8]   Kendall, F.E., Heidelberger, M. and Dawson, M.H. (1937) A serologically inactive polysaccharide elaborated by mucoid strains of group A hemolytic streptococcus. Journal of Biological Chemistry, 118, 61-69.

[9]   Carter, G.R. and Annau, E. (1953) Isolation of capsular polysaccharides for colonial variants of Pasteurella multocida. American Journal of Veterinary Research, 14, 475-478.

[10]   MacLennan, A.P. (1956) The production of capsules, hyaluronic acid and hyaluronidase by 25 strains of group C streptococci. Journal of General Microbiology, 15, 485-491. http://dx.doi.org/10.1099/00221287-15-3-485

[11]   Thonard, J.C., Migliore, S.A. and Blustein, R. (1964) Isolation of hyaluronic acid from broth cultures of streptococci. Journal of Biological Chemistry, 239, 726-728.

[12]   Armstrong, D.C. and Johns, M.R. (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. Metabolic Engineering, 10, 24-32. http://dx.doi.org/10.1016/j.ymben.2007.09.001

[17]   Mao, Z. and Chen, R.R. (2007) Recombinant synthesis of hyaluronan by agrobacterium sp. Biotechnology Progress, 23, 1038-1042.

[18]   Mao, Z., Shin, H.-D. and Chen, R. (2009) A recombinant E. coli bioprocess for hyaluronan synthesis. Applied Microbiology and Biotechnology, 84, 63-69. http://dx.doi.org/10.1007/s00253-009-1963-2

[19]   Johns, M.R., Goh, L.-T. and Oeggerli, A. (1994) Effect of pH, agitation and aeration on hyaluronic acid production by Streptococcus zooepidemicus. Biotechnology Letters, 16, 507-512. http://dx.doi.org/10.1007/BF01023334

[20]   Huang, W.-C., Chen, S.-J. and Chen, T.-L. (2006) The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation. Biochemical Engineering Journal, 32, 239-243. http://dx.doi.org/10.1016/j.bej.2006.10.011

[21]   Kim, J.-H., et al. (1996) Selection of a Streptococcus equi mutant and optimization of culture conditions for the production of high molecular weight hyaluronic acid. Enzyme and Microbial Technology, 19, 440-445. http://dx.doi.org/10.1016/S0141-0229(96)00019-1

[22]   Chen, W.Y., et al. (2009) Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus. Journal of Biological Chemistry, 284, 18007-18014. http://dx.doi.org/10.1074/jbc.M109.011999

[23]   Krupa, J.C., et al. (2007) Quantitative continuous assay for hyaluronan synthase. Analytical Biochemistry, 361, 218-225. http://dx.doi.org/10.1016/j.ab.2006.11.011

[24]   Pummill, P.E., Achyuthan, A.M. and DeAngelis, P.L. (1998) Enzymological characterization of recombinant Xenopus DG42, a vertebrate hyaluronan synthase. Journal of Biological Chemistry, 273, 4976-4981. http://dx.doi.org/10.1074/jbc.273.9.4976

[25]   Itano, N., et al. (1999) Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. Journal of Biological Chemistry, 274, 25085-25092. http://dx.doi.org/10.1074/jbc.274.35.25085

[26]   Tlapak-Simmons, V.L., et al. (1999) Kinetic characterization of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis. Journal of Biological Chemistry, 274, 4246-4253. http://dx.doi.org/10.1074/jbc.274.7.4246

[27]   DeAngelis, P.L. (1999) Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase. Journal of Biological Chemistry, 274, 26557-26562. http://dx.doi.org/10.1074/jbc.274.37.26557

[28]   Jing, W. and DeAngelis, P.L. (2004) Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. Journal of Biological Chemistry, 279, 42345- 42349. http://dx.doi.org/10.1074/jbc.M402744200

[29]   Osawa, T., et al. (2009) Crystal structure of chondroitin polymerase from Escherichia coli K4. Biochemical and Biophysical Research Communications, 378, 10-14. http://dx.doi.org/10.1016/j.bbrc.2008.08.121

[30]   Jing, W. and DeAngelis, P.L. (2000) Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: Two active sites exist in one polypeptide. Glycobiology, 10, 883-889. http://dx.doi.org/10.1093/glycob/10.9.883

[31]   Fitzgerald, D.K., et al. (1970) Enzymic assay for galactosyl transferase activity of lactose synthetase and [alpha]lactalbumin in purified and crude systems. Analytical Biochemistry, 36, 43-61. http://dx.doi.org/10.1016/0003-2697(70)90330-1

[32]   Gosselin, S., et al. (1994) A continuous spectrophotometric assay for glycosyltransferases. Analytical Biochemistry, 220, 92-97. http://dx.doi.org/10.1006/abio.1994.1303

[33]   Kooy, F.K., et al. (2009) Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis. Analytical Biochemistry, 384, 329-336. http://dx.doi.org/10.1016/j.ab.2008.09.042

[34]   Cornish-Bowden, A. (1995) Fundamentals of enzyme kinetics. Portland Press Ltd., London.

[35]   Cook, P.F. and Cleland, W.W. (2007) Enzyme kinetics and mechanism. Garland Science, London.

[36]   De Levie, R. (2004) Macros for least-squares & for the propagation of imprecision, in advanced excel for scientific data analysis. Oxford University Press, New York.

[37]   Van Boekel, M.A.J.S. (2010) Kinetic modeling of reactions in foods. CRC Press, Boca Raton.

[38]   Motulsky, H. and Christopoulos, A. (2003) Fitting models to biological data using linear and nonlinear regression: A practical guide to curve fitting. GraphPad Software Inc., San Diego.

[39]   Williams, K.J., Halkes, K.M., Kamerling, J.P. and DeAngelis, P.L. (2006) Critical elements of oligosaccharide acceptor substrates for the Pasteurella multocida hyaluronan synthase. Journal of Biological Chemistry, 281, 5391- 5397. http://dx.doi.org/10.1074/jbc.M510439200

[40]   Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993) PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystalography, 26, 283-291. http://dx.doi.org/10.1107/S0021889892009944

[41]   Sippl, M.J. (1993) Recognition of errors in three-dimensional structures of proteins. Proteins: Structure, Function, and Genetics, 17, 355-362. http://dx.doi.org/10.1002/prot.340170404

[42]   Trott, O. and Olson, A.J. (2009) AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry, 31, 455-461.

[43]   Holm, L. and Sander, C. (1996) Mapping the protein universe. Science, 273, 595-602. http://dx.doi.org/10.1126/science.273.5275.595

[44]   DeAngelis, P.L. (1996) Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase. Biochemistry, 35, 9768-9771. http://dx.doi.org/10.1021/bi960154k

[45]   Tlapak-Simmons, V.L., Baron, C.A. and Weigel, P.H. (2004) Characterization of the purified hyaluronan synthase from Streptococcus equisimilis. Biochemistry, 43, 9234-9242. http://dx.doi.org/10.1021/bi049468v

[46]   Yoshida, M., Itano, N., Yamada, Y. and Kimata, K. (2000) In Vitro synthesis of hyaluronan by a single protein derived from mouse HAS1 Gene and characterization of amino acid residues essential for the activity. Journal of Biological Chemistry, 275, 497-506. http://dx.doi.org/10.1074/jbc.275.1.497

[47]   Kumari, K. and Weigel, P.H. (1997) Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from Group C Streptococcus equisimilis. Journal of Biological Chemistry, 272, 32539-32546. http://dx.doi.org/10.1074/jbc.272.51.32539

[48]   Eisenthal, R., Danson, M.J. and Hough, D.W. (2007) Catalytic efficiency and kcat/KM: A useful comparator? Trends in Biotechnology, 25, 247-249. http://dx.doi.org/10.1016/j.tibtech.2007.03.010

[49]   Breton, C., Snajdrová, L., Jeanneau, C., Koca, J. and Imberty, A. (2006) Structures and mechanisms of glycosyl-transferases. Glycobiology, 16, 29R-37R. http://dx.doi.org/10.1093/glycob/cwj016

[50]   Fritz, T.A., Raman, J. and Tabak, L.A. (2006) Dynamic association between the catalytic and lectin domains of human UDP-GalNAc: Polypeptide a-N-acetylgalactosami- nyltransferase-2. Journal of Biological Chemistry, 281, 8613-8619. http://dx.doi.org/10.1074/jbc.M513590200

[51]   Pedersen, L.C., Tsuchida, K., Kitagawa, H., Sugahara, K., Darden, T.A. and Negishi, M. (2000) Heparan/Chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I. Journal of Biological Chemistry, 275, 34580-34585. http://dx.doi.org/10.1074/jbc.M007399200

[52]   Kakuda, S., Shiba, T., Ishiguro, M., Tagawa, H., Oka, S., Kajihara, Y., Kawasaki, T., Wakatsuki, S. and Kato, R. (2004) Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1. Journal of Biological Chemistry, 279, 22693- 22703. http://dx.doi.org/10.1074/jbc.M400622200

[53]   Ramasamy, V., Ramakrishnana, B., Boeggeman, E., Ratner, D.M., Seeberger, P.H. and Qasba, P.K. (2005) Oligosaccharide preferences of β1,4-galactosyltransferase-I: Crystal structures of Met340His mutant of human β1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the Nglycan moiety. Journal of Molecular Biology, 353, 53-67. http://dx.doi.org/10.1016/j.jmb.2005.07.050

[54]   Pedersen, L.C., Dong, J., Taniguchi, F., Kitagawa, H., Krahn, J.M., Pedersen, L.G., Sugahara, K. and Negishi, M. (2003) Crystal structure of an 1,4-N-acetylhexosami- nyltransferase (EXTL2), a member of the exostosin gene family involved in heparan sulfate biosynthesis. Journal of Biological Chemistry, 278, 14420-14428. http://dx.doi.org/10.1074/jbc.M210532200

[55]   Zhang, Y., Swaminathan, G.J., Deshpande, A., Boix, E., Natesh, R., Xie, Z.H., Acharya, K.R. and Brew, K. (2003) Roles of individual enzyme—Substrate interactions by α-1,3-galactosyltransferase in catalysis and specificity. Biochemistry, 42, 13512-13521. http://dx.doi.org/10.1021/bi035430r

[56]   Patenaude, S.I., Seto, N.O., Borisova, S.N., Szpacenko, A., Marcus, S.L., Palcic, M.M. and Evans, S.V. (2002) The structural basis for specificity in human ABO(H) blood group biosynthesis. Nature Structural Biology, 9, 685-690. http://dx.doi.org/10.1038/nsb832

[57]   Persson, K., Ly, H.D., Dieckelmann, M., Wakarchuk, W.W., Withers, S.G. and Strynadka, N.C.J. (2001) Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nature Structural Biology, 8, 166-175. http://dx.doi.org/10.1038/84168

[58]   Breton, C., Bettler, E., Joziasse, D.H., Geremia, R.A. and Imberty, A. (1998) Sequence-Function relationships of prokaryotic and eukaryotic galactosyltransferases. Journal of Biochemistry, 123, 1000-1009. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022035

[59]   Tarbouriech, N., Charnock, S.J. and Davies, G.J. (2001) Three-Dimensional structures of the Mn and Mg dTDP complexes of the family GT-2 glycosyltransferase SpsA: A comparison with related NDP-sugar glycosyltransferases. Journal of Molecular Biology, 314, 655-661. http://dx.doi.org/10.1006/jmbi.2001.5159

[60]   Jing, W. and DeAngelis, P.L. (2003) Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida. Glycobiology, 13, 661-671. http://dx.doi.org/10.1093/glycob/cwg085

[61]   Pedersen, L.C., Darden, T.A. and Negishi. M. (2002) Crystal structure of b1,3-glucuronyltransferase I in complex with active donor substrate UDP-GlcUA. Journal of Biological Chemistry, 277, 21869-21873. http://dx.doi.org/10.1074/jbc.M112343200

[62]   Gastinel, L.N., Bignon, C., Misra, A.K., Hindsgaul, O., Shaper, J.H. and Joziass, D.H. (2001), Bovine a1,3-galacto-syltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases. EMBO Journal, 20, 638-649. http://dx.doi.org/10.1093/emboj/20.4.638

[63]   Ramakrishnan, B., Boeggeman, E. and Qasba, P.K. (2004) Effect of the Met344His mutation on the conformational dynamics of bovine b-1,4-galactosyltransferase: Crystal structure of the Met344His mutant in complex with chitobiose. Biochemistry, 43, 12513-12522.

[64]   Negishi, M., Donga, J., Dardenb, T.A., Pedersenb, L.G. and Pedersen, L.C. (2003) Glucosaminylglycan biosynthesis: What we can learn from the X-ray crystal structures of glycosyltransferases GlcAT1 and EXTL2. Biochemical and Biophysical Research Communications, 303, 393-398. http://dx.doi.org/10.1016/S0006-291X(03)00356-5

[65]   Fondeur-Gelinotte, M., et al. (2007) Molecular basis for acceptor substrate specificity of the human β1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively. Glycobiology, 17, 857-867. http://dx.doi.org/10.1093/glycob/cwm055

[66]   Zhang, Y., Deshpande, A., Xie, Z.H., Natesh, R., Acharya, K.R. and Brew, K. (2004) Roles of active site tryptophans in substrate binding and catalysis by α-1,3 galactosyl- transferase. Glycobiology, 14, 1295-1302. http://dx.doi.org/10.1093/glycob/cwh119

[67]   Mulders, K.J.M. and Beeftink, H.H. (2013) Chain length distribution and kinetic characteristics of an enzymaticcally produced polymer. e-Polymers, 24, 1-12.