The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (ϳ10 2-4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.
Glycosaminoglycans (GAGs),2 polysaccharides containing a hexosamine as part of their repeat unit, serve essential biological functions in vertebrates, including adhesion, modulation of motility and proliferation, and coagulation (1-8). Certain pathogenic bacteria camouflage themselves with GAGs to increase virulence and enhance infection in their animal hosts. These capsular polysaccharides serve as molecular camouflage as well as a means to potentially hijack host functions (9). The backbones of the vertebrate GAGs, HA (-1,4-GlcUA-1,3-GlcNAc-), chondroitin sulfate (-1,4-GlcUA-1,3-GalNAc-), heparan sulfate (␣1,4-GlcUA-1,4-GlcNAc-), heparin (-␣1,4-IdoUA-␣ 1,4-GlcNAc-), and keratan sulfate (-1,3-Gal-1,4-GlcNAc-), are sulfated except for HA. The bacterial GAG polymers are not known to be sulfated. The enzymes involved in the biosynthesis of all the GAG backbones, except for keratan, have been molecularly cloned.Certain bacterial enzymes, including the Gram-negative Pasteurella multocida type A hyaluronan synthase, PmHAS, and the type F chondroitin synthase, PmCS, are particularly amenable to study because of their two active center architecture (10, 11), their ability to polymerize long chains in vitro (12), and their ability to elongate exogenously supplied acceptor oligosaccharides in vitro (13,14). The Escherichia ...