PROCESSING AND HYDROLYTIC MECHANISM OF THE CGKA-ENCODED KAPPA-CARRAGEENASE OF ALTEROMONAS-CARRAGEENOVORA

The cgkA gene of Alteromonas carrageenovora encodes a kappa-carrageena se with a predicted mass of 44212 Da, much larger than the 35 kDa esti mated from SDS/PAGE of the protein purified from culture supernatants. Immunoblotting experiments showed the presence of a protein of 44 +/- 2 kDa in both native and recombinant bacterial intracellular extracts , suggesting that the kappa-carageenase is produced as a preproprotein which undergoes proteolytic processing twice during secretion. To det ermine the exact site of C-terminal cleavage, the precise mass of the purified extracellular kappa-carrageenase was measured by electrospray -ionization/mass spectrometry and found to be 31 741 +/- 3 Da. The mat ure kappa-carrageenase of A. carrageenovora thus appears to be compose d of 275 amino acids, from residue Ala26 to residue Asn301 of the cgkA gene product. To assess the molecular mechanism of this member of fam ily 16 of glycosyl hydrolases, hydrolysis of neocarrahexaitol by the k appa-carrageenase was monitored by gel filtration chromatography and C -13-NMR. Results show that neocarrabiitol and beta-neocarratetraose ar e initially formed, demonstrating that the enzyme operates with a mole cular mechanism retaining the anomeric configuration. Consistent with this result, the enzyme was also shown to be able to catalyze transgly cosylation.