Fig. 35: GPI-anchored substrates are delivered by Dfg5 enzymes to the fungal cell wall using a covalent two-step mechanism. a) Schematic representation of the principal transglycosylation procedure catalysed by ascomycetal GH76 enzymes. GPI-AP: GPI-anchored protein, GPI-CWP: GPI-cell wall protein. b) In-crystallo glycan fragment screening revealed the GPI-core glycan within the binding pocket of Dfg5 from glycan subsites -3 to +1 with the scissile bond above the active site DD-motif. The acceptor glycan fragment laminaribiose is shown with the non-reducing end towards the active site sitting at glycan subsites +1 to +2. The theoretical GPI-CWP and PI-lipid positions are indicated. c) A snapshot from MD simulations shows the membrane-bound Dfg5 from C. thermophilum bound to a GPI-anchored w-peptide. d) Model of the membrane-to- wall transfer as catalysed by Dfg5 enzymes. TM: transmembrane anchor, PM: plasma membrane.
whereas a sequence similarity analysis supports a distinct and isofunctional subfamily for ascomycetal Dfg5 enzymes . With high- molar, in-crystallo glycan fragment screening, a novel approach to reconstruct complex glycans that otherwise escape characterisation due to difficult chemical synthesis was introduced. By characterising CtDfg5 fragment co-crystals at beamlines ID23-1, ID23-2, and ID29 (Figure 35b), it was possible to reassemble the whole GPI-core glycan within the binding pocket of CtDfg5 from glycan binding sites +1 to -3. These findings were validated by MD simulations using the observed GPI-core conformation as a starting point to reconstruct a dynamically stable arrangement of Dfg5 anchored to plasma membrane via its C-terminal transmembrane helix when complexed to its cognate substrate a GPI-anchored w-peptide (Figure 35c). The acceptor bound state further revealed the binding of laminaribiose (β-1,3 linked glucoses) with the non-reducing end at glycan binding
site +1, similar to the to-be-cleaved glucosamine from the GPI-core (Figure 35b).
Based on these findings, a two-step transfer mechanism was proposed (Figure 35d). The first step includes GPI-anchor binding and lysis of the mannose-α1,4-glucosamine glycosidic bond. By doing so, the phosphatidylinositol-glucosamine lipid remains in the plasma membrane, while a covalent enzyme-glycosyl intermediate is established with the remainder. In the second step, the non-reducing end of β1,3-glucan binds to the unoccupied glycan subsite +1 previously occupied by the glucosamine moiety. There, it is activated by the general acid/base residue D135, accordingly breaking the covalent intermediate while reforming a new α1,4-glycosidic bond between mannose and the terminal glucose.
By this mechanism, the GPI-anchored membrane protein becomes a GPI-anchored cell wall protein. Interestingly, the catalytically