Antifungal activities of LysM-domain multimers and their fusion chitinases

Abstract

PrChiA is an antifungal chitinase obtained from Pteris ryukyuensis, a fern plant. It consists of two N-terminal lysin motif (LysM) domains and a C-terminal catalytic domain of glycoside hydrolase family 18. Previous studies have shown that the deletion of LysM domains or loss of hydrolytic activity causes the loss of the antifungal activity of chitinases. In this study, we produced LysM-domain multimers (LysMn, n = 2–5) and the respective multimer fusion chitinases (LysMn-Cat, n = 1–4), and characterized their enzymatic and antifungal properties. LysMn and LysMn-Cat showed a higher affinity to insoluble chitin than single LysM domain and single catalytic domain alone, respectively. LysMn-Cat hydrolyzed insoluble chitin more efficiently than the catalytic domain alone. Surprisingly, LysMn showed antifungal activity without chitinolytic activity. Further, LysMn-Cat exhibited a stronger antifungal activity than LysMn. Microscopic observation revealed that LysMn attacked only the tips of the fungal hyphae; LysMn-Cat attacked not only the tips, but also the lateral walls around the septa of the fungal hyphae. It is suggested that the LysMn act on the growing point of the hyphal tip through their chitin-binding ability and that the LysMn-Cat act on not only the hyphal tips, but also on the lateral walls through their chitin-hydrolyzing and -binding activities.