Pasteurella GAG synthases

An interesting feature of the Pasteurella GAG synthases is that they appear to elongate the acceptors in a non-processive fashion in vitro with chain release between sugar addition steps. As noted later, this property facilitates chemoenzymatic synthesis reactions in vitro. 

The Pasteurella GAG synthases add sugars onto the non-reducing terminus of an existing GAG chain in a rapid fashion, but if no acceptor is present, the enzymes will spontaneously initiate new chain formation de novo. This initiation rate is slower than the elongation rate. The DeAngelis Laboratory found it is possible to accelerate and to synchronize GAG chain production by adding an acceptor molecule to the reaction mixture. The synchronized non-processive polymerization of all chains in concert results in the production of product with... 

The native Pasteurella and the recombinant Escherichia co//-derived preparations of the various microbial Pasteurella GAG synthases rapidly form long polymer chains in vitro (2). The sugar transfer specificity of the native-sequence enzymes is exquisite and only the authentic sugars are incorporated into polymer products. For example, the native synthase enzymes do not utilize significantly the C4 epimer precursors in comparison to the natural UDP-sugars. 

The native bacterial GAG glycosyltransferase polypeptides are associated with the cell membranes this localization makes sense with respect to synthesis of polysaccharide molecules destined for the cell surface. The first Pasteurella GAG synthase to be identified was the 972-residue HA synthase from Type A strains, pmHAS (Table I). This single polypeptide transfers both sugars, GlcNAc and GlcUA, to form the HA disaccharide repeat (i). 

We recently found that the various Pasteurella GAG synthases can also use non-cognate acceptor molecules. In the example shown in Fig. 2, we added a HA chain onto an existing chondroitin sulfate chain using pmHAS (P). This particular family of hybrid molecules may be useful as artificial proteoglycans for tissue engineering that do not contain the protein component of the natural cartilage proteoglycans. 

We have developed the chemoenzymatic synthesis of monodisperse GAG oligosaccharides (10). Potential medical applications for HA oligosaccharides (n = -3-10) include killing cancerous tumors and enhancing wound vascularization. The Pasteurella HA synthase, a polymerizing enzyme that... 

Typically, the native sequence GAG synthases will only transfer the authentic UDP-sugars to produce the natural polymer. However, we have now created new enzymes that can make novel polymers that are not known to exist in Nature. We used a chimeric genetic modification approach to map out the Gal/Glc specificity for UDP-hexosamines of the Pasteurella pmHAS [HA... 

Versatile, malleable Pasteurella synthases have been harnessed as useful catalysts for the creation of a variety of defmed GAG or GAG-like polymers ranging in size from small oligosaccharides to huge polysaccharides. These materials should be useful for a wide spectrum of potential biomedical products for use in the areas of cancer, coagulation, infection, tissue engineering, drug delivery, surgery, and viscoelastic supplementation. 

---