The production of unsaturated uronides by bacterial hyaluronidases.

Hyaluronic acid (HA)’ is a polysaccharide composed of equimolar amounts of n-glucuronic acid and N-acetylglucosamine. The basic repeating unit of the straight chain polymer is the disaccharide, N-acetylhyalobiuranic acid (3P-D-glucuronopyranosyl-N-acetylglucosamine (3)). The polysaccharide is hydrolyzed by enzymes from various sources such as mammalian testis, bacteria, snake venom, and others. It has been shown previously that only the glucosaminidic linkages of the polymer are hydrolyzed by both testicular (4) and pneumococcal hyaluronidases (5). As the rate and extent of hydrolysis were found to be much greater with the bacterial hyaluronidase, it was postulated that with this enzyme a disaccharide and with the testis enzyme a tetrasaccharide were produced (5). It was reported subsequently that the end-product of testicular hyaluronidase digestion was a mixture of oligosaccharides containing tetrasaccharide as a major component (6). It will be seen here that enzymes obtained from various bacterial sources degrade HA to the same end-product, a disaccharide, which is different from N-acetylhyalobiuronic acid (N-acetyl-HBA), the repeating unit of the polymer. This difference will be shown to be due to the modifying action of the bacterial enzymes during the cleavage process equivalent to the elimination of 1 molecule of water from the uranic acid portion of the repeating unit, resulting in the formation of a disaccharide containing a 4,5-unsaturated uranic acid, a new type of sugar acid. To our knowledge, no other example of the appearance of unsaturation during the enzymic depolymerization of a polysaccharide has been observed.