Characterizing the role of an α1,6‐mannanase needed for Neurospora crassa cell wall biogenesis

Abstract

Cell wall components such as glucans, chitins and mannans are needed for fungal virulence. The dfg‐5 gene encodes a GH‐76 α‐ 1,6 mannanase that is needed to cleave the α‐1,6‐mannose backbone of fungal N‐linked oligosaccharide‐associated galactomannans. The processed galactomannan is then used as substrate by GH‐72 lichenin transferases, that cross‐link it into the cell wall. However the mechanism of how DFG‐5 processes the galactomannan structure remains unclear. Here, we characterize the DFG‐5 protein. A pair of adjacent aspartic acid (D) or glutamic acid (E) residues are normally found at the active sites of GH‐76 family glycosylhydrolases. We used site directed mutagenesis to mutate 7 conserved aspartate and glutamate residues in DFG‐5 protein. Complementation analysis demonstrates that D76, D116, D117, and E130 mutations renders DFG‐5 inactive. 3D Modeling of DFG5 protein strongly suggests that D116 and D117 define the active site based on homology with other characterized glycosylhydrolases. Modeling of DFG‐5 mannanase shows it has a second groove containing D76 and E130. To further characterize DFG‐5, we generated a HIS6 tagged version of the protein and demonstrated that it complemented a dfg‐5 gene deletion. A number of glycoproteins co‐purify with the HIS6‐tagged DFG‐5 and these co‐purifying glycoproteins require galactomannan for DFG‐5 binding. Three known cell wall glycoproteins co‐purify with DFG‐5 mannanse.Support or Funding InformationFunding for the study is provided by the UB Foundation.