ι-Carrageenases constitute a novel family of glycoside hydrolases, unrelated to that of κ-carrageenases

Abstract

ι-Carrageenases are polysaccharide hydrolases that cleave the β-1,4 linkages between the D-galactose-4-sulfate and 3,6-anhydro-D-galactose-2-sulfate residues in the red algal galactans known as ι-carrageenans. We report here on the purification of ι-carrageenase activity from the marine bacterium Zobellia galactanovorans and on the characterization of ι-carrageenase structural genes. Genomic libraries from this latter bacterium as well as from Alteromonas fortis were functionally screened for the presence of ι-carrageenase+ clones. The Z. galactanovorans and A. fortis ι-carrageenase genes encode homologous proteins of 53.4 and 54.8 kDa, respectively. Based on hydrophobic cluster analysis and on the 1H NMR monitoring of the products of the over-expressed A. fortis ι-carrageenase, these enzymes appear to form a new family of glycoside hydrolases, un-related to that of κ-carrageenases and with an inverting mechanism of hydrolysis. They both feature a 45-amino acid-long N-terminal segment with sequence similarity to the N-terminal region of several other polysaccharidases. In those for which a three-dimensional structure is available, this conspicuous segment, also deemed 'glycanase motif' (Chua, J. E. H., Manning, P. A., and Morona, R. (1999) MicroBiology (Reading) 145, 1649-1659), corresponds to a strand-helix-strand 'cap' that covers the N-terminal end of a common, right-handed β-helical fold.