Pasteurella synthases

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. 

Recent work on the biosynthesis of HA in Pasteurella multocida has shown that in this organism HA is elongated by addition of sugar residues to the non-reducing end [40]. The Pasteurella synthase has a distinctly different amino acid sequence than other HA synthases and it may be that there exists two different pathways to make HA from the same UDP-sugars. However, the observation by DeAngelis [40] may necessitate a reanalysis of the proposed mechanism in streptococci and eukaryotic cells. 

HA synthase from Streptococcus equisimilis was employed for milligram-scale synthesis of HA (109). In this reaction system, UDP-sugars (UDP-GlcA and UDP-GlcNAc) were effectively regenerated by the catalyses of several enzymes, synthetic HA was produced in 90% yield. In addition, mutated HA synthase from Type A Pasteurella multocida (PmHAS 1-703 aa) was recently used for the stepwise synthesis of HA, which has a monodispersed molecular mass of up to 20 sugar units (110). 

DeAngelis PL, White CL. Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D. J. Biol. Chem 2002 277 7209-7213. 

A unique HAS enzyme occurs in the virus that infects an algae Pasteurella multocida (pmHAS) that is quite different from all other HA synthases [94]. This is in a class by itself and will not be considered further. 

The 617-residue Type D Pasteurella heparosan synthase, PmHSl, and the 651-residue Type A,D, and F Pasteurella cryptic heparosan synthase, PmHS2, are not very similar at the protein level to either PmHAS or PmCS. Both of these enzymes produce unsulfated heparosan chains. The PmHS enzyme, however, resembles a fusion of the E. coli K5 KfiA and KfiC proteins ... 

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). 

The Pasteurella chondroitin synthase, pmCS, contains separate GalNAc-transferase (a slightly mutated version of the GlcNAc-site of pmHAS) and GlcUA-transferase sites. The pmHS domains are still be investigated. These single-action enzymes are useful catalysts for the preparation of defined oligosaccharides described later. 

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... 

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