Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protect <i>aku</i>BGL activity.

Abstract

The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator <i>Aplysia kurodai</i>, which uses β-glucosidase (<i>aku</i>BGL) to digest the laminarin in algae into glucose. Moreover, <i>A. kurodai</i> employs <i>Eisenia</i> hydrolysis-enhancing protein (EHEP) as an offense to protect <i>aku</i>BGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of the apo and tannic acid (TNA, a phlorotannin analog) bound forms of EHEP, as well as the apo <i>aku</i>BGL. EHEP consisted of three peritrophin-A domains arranged in a triangular shape and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP-TNA led us to find that EHEP can be resolubilized from phlorotannin precipitation at an alkaline pH, which reflects a requirement in the digestive tract. <i>aku</i>BGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits <i>aku</i>BGL by occupying the substrate-binding pocket, and EHEP protects <i>aku</i>BGL against this inhibition by binding with phlorotannin to free the <i>aku</i>BGL pocket.