Processing and hydrolytic mechanism of the cgkA-encoded kappa-carrageenase of Alteromonas carrageenovora.
The cgkA gene of Alteromonas carrageenovora encodes a kappa-carrageenase with a predicted mass of 44212 Da, much larger than the 35 kDa estimated 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-carrageenase is produced as a preproprotein which undergoes proteolytic processing twice during secretion. To determine 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 mature kappa-carrageenase of A. carrageenovora thus appears to be composed of 275 amino acids, from residue Ala26 to residue Asn301 of the cgkA gene product. To assess the molecular mechanism of this member of family 16 of glycosyl hydrolases, hydrolysis of neocarrahexaitol by the kappa-carrageenase was monitored by gel filtration chromatography and 13C-NMR. Results show that neocarrabiitol and beta-neocarratetraose are initially formed, demonstrating that the enzyme operates with a molecular mechanism retaining the anomeric configuration. Consistent with this result, the enzyme was also shown to be able to catalyze transglycosylation.
Potin
P
P
Centre d'Etudes d'Océanographie et de Biologie Marine, CNRS UPR 4601, Roscoff, France.
Richard
C
C
Barbeyron
T
T
Henrissat
B
B
Gey
C
C
Petillot
Y
Y
Forest
E
E
Dideberg
O
O
Rochas
C
C
Kloareg
B
B
D001426:Bacterial Proteins; D015153:Blotting, Western; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D011499:Protein Processing, Post-Translational; D013237:Stereoisomerism
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C019846:kappa-carrageenase protein, Alteromonadaceae