Probing the structure of glucan lyases – the lytic members of GH31 - by sequence analysis, circular dichroism and proteolysis

Glucan lyase (GL) is a polysaccharide lyase with unique characteristics. It is involved in an alternative pathway for the degradation of alpha-glucans, the anhydrofructose pathway. Sequence similarity suggests that this lytic enzyme belongs to glycoside hydrolase family 31, for which until very recently no structural representative was available. In the present study, the GLs have been analysed by bioinformatics, and experimental data have been obtained for two isozymes from the red alga Gracilariopsis lemaneiformis by circular dichrosim and limited proteolysis. Based on these results, the GLs are predicted to have a central catalytic domain with (beta/alpha)(8) structure flanked by beta-rich domains at the N- and C-termini. The GLs were found to be surprisingly resistant to proteolytic degradation, requiring relatively high protease concentrations and long incubation times for cleavage to occur. Two cleavage sites have been identified in the N-terminal part of the protein, while the central domain and the C-terminal region do not seem to be susceptible to proteolytic attack. These results suggest that GLs are compact in structure, unlike many carbohydrate-modifying enzymes consisting of modules connected by long flexible linkers.