MULTIPLE ENDOGLYCOSIDASE-F ACTIVITIES EXPRESSED BY FLAVOBACTERIUM-MENINGOSEPTICUM ENDOGLYCOSIDASES-F2 AND ENDOGLYCOSIDASES-F3 - MOLECULAR-CLONING, PRIMARY SEQUENCE, AND ENZYME EXPRESSION

The genes for Flavobacterium meningosepticum Endo (endoglycosidase) F2 and Endo F3 were cloned, and their nucleotide sequences were determin ed. The deduced amino acid sequences were verified independently to a large extent by direct peptide microsequencing of 66 and 84% of native Endo F2 and Endo F3, respectively. Structurally, the Endo F2 and Endo F3 genes code for a typically long leader sequence of 45 and 39 amino acids, respectively, and, in both cases, a mature protein of 290 amin o acids. Comparative structural analysis demonstrated minimum overall homology (15-30%) between Endo F1, Endo F2, and Endo F3, but revealed distinct clusters of identical residues distributed throughout the ent ire sequence, which represent motifs for binding and hydrolysis of bet a1,4-di-N-acetylchitobiosyl linkages in complex carbohydrates. The mob ility of native Endo F2 and Endo F3 on SDS-polyacrylamide gel electrop horesis, unlike Endo F1, did not correlate with the molecular weights determined from the coding region of the corresponding genes. Mass spe ctrometry confirmed that Endo F2 and Endo F3 were heterogeneous and co ntained approximately 4000 and 1200 daltons of mass not accounted for in the gene structure. We presume that Endo F2 and Endo F3 are variabl y post-translationally modified during secretion by possible linkage t o the hydroxyl of serine.