IMMUNOCHEMICAL CONFIRMATION OF THE PRIMARY STRUCTURE OF STREPTOCOCCAL HYALURONAN SYNTHASE AND SYNTHESIS OF HIGH-MOLECULAR-WEIGHT PRODUCT BY THE RECOMBINANT ENZYME

We have recently identified and cloned the gene for hyaluronan (HA) synthase, hasA, from group A Streptococci [DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184]. We have now generated two polyclonal monospecific antibodies against synthetic peptides corresponding to portions of the deduced protein. Both antibodies recognize a protein with an apparent molecular weight of 42 000 either from wild-type Streptococcus pyogenes or from Escherichia coli containing the cloned gene on a plasmid. Immobilized affinity-purified antibody depleted HA synthase activity from functional detergent extracts of streptococcal membranes in a specific fashion. The immobilized protein displayed HA synthase activity, and HasA was the major bound polypeptide. The recombinant HA synthase behaves identically to that from Streptococci, with respect to sugar nucleotide specificity and polysaccharide production. Only the authentic sugar nucleotides UDP-glucuronic acid and UDP-N-acetylglucosamine support HA polymerization. The recombinant enzyme elongates HA in a processive manner and rapidly produces polymers on the order of greater than or equal to 5 x 10(6) Da at rates of about 10-30 monosaccharides/s at three times the apparent K-m of substrates.