Characterizing the role of assembly on the function of chorismate synthase in Neurospora crassa

Chorismate synthase is the seventh enzyme in the shikimate pathway and has been identified as a potential anti-fungal target. Important differences exist between fungal and bacterial chorismate synthases, but almost no structural data exists for chorismate synthase in fungi. We have identified the chorismate synthase from Neurospora crassa as a good homologue for pathogenic fungi like Histoplasma capsulatum because of the high sequence identity. To generate protein for crystallization, chorismate synthase from Neurospora crassa was expressed in E. coli using a modified pET expression vector that included an N-terminal histidine tag. After unsuccessful attempts to cleave the tag, analytical ultracentrifugation sedimentation velocity experiments demonstrated that the protein sample was a heterologous mixture of dimer, tetramer, and high-molecular weight aggregates. Site-directed mutagenesis was used to remove the N-terminal His tag and TEV cleavage site and then to insert a C-terminal affinity tag. By sedimentation velocity it was demonstrated that the C-terminal tagged construct assembled as a tetramer only. Using a coupled-assay it was demonstrated that only the tetrameric construct has the flavin reductase capabilities associated with bifunctional chorismate synthases. These data provide evidence that tetrameric assembly is blocked by the presence of an N-terminal tag, which disrupts flavin reductase activity. Our findings will guide best practices for designing expression vectors and purification techniques for ongoing work to pursue structural studies of fungal chorismate synthases.