The collagenase secreted by Grimontia hollisae strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C-terminal segment that includes the bacterial pre-peptidase C-terminal domain. Here, we produced a recombinant C-terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen-binding domains (CBDs) derived from Hathewaya histolytica (Clostridium histolyticum) collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C-terminal segment binds to collagen only when the collagen is in its triple-helical conformation. Moreover, the C-terminal segment and the CBDs from H. histolytica have comparable characteristics, including binding affinity to type I collagen, substrate spectrum, and binding conditions with respect to salt concentration and pH. However, the C-terminal segment has a completely different primary structure from those of the CBDs from H. histolytica. As regards secondary structure, in silico prediction indicates that the C-terminal segment may be homologous to those in CBDs from H. histolytica. Furthermore, we performed collagenase assays using fluorescein isothiocyanate-labeled type I collagen to show that the C-terminal segment positively contributes to the collagenolytic activity of the 74 kDa collagenase from G. hollisae.
Keywords: Grimontia hollisae; PPC domain; bacterial collagenase; metallopeptidase M9 subfamily A; recombinant protein; triple‐helical conformation.