Structure-function analysis of Cmu1, the secreted chorismate mutase from Ustilago maydis
The basidiomycete fungus Ustilago maydis is the causative agent for smut disease of maize (Zea mays). More than 400 putative secreted proteins are encoded in the genome of U. maydis. The secreted chorismate mutase Cmu1 of U. maydis is such a translocated virulence promoting effector. The chorismate...
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|Zusammenfassung:||The basidiomycete fungus Ustilago maydis is the causative agent for smut disease of maize (Zea mays). More than 400 putative secreted proteins are encoded in the genome of U. maydis. The secreted chorismate mutase Cmu1 of U. maydis is such a translocated virulence promoting effector. The chorismate mutase activity of Cmu1 in the cytosol is proposed to lower the chorismate levels in the chloroplast where it would serve as precursor for the biosynthesis of the plant defense hormone salicylic acid (SA). The crystal structure of Cmu1 revealed several unique features in comparison to the cytoplasmic chorismate mutase Aro7p of Saccharomyces cerevisiae, including a surface exposed acidic patch, a disulfide bond, a putative fatty acid binding site and a loop region. This thesis shows, that site-directed mutagenesis affecting the acidic patch, the disulfide bond and the fatty acid binding site results in functional mutant proteins that can complement the virulence phenotype of CL13Δcmu1 strains. Wildtype Cmu1 protein purified after heterologous expression in E. coli followed a Michaelis-Menten kinetic in a chorismase mutase activity assay. Mutations in the fatty acid binding site did not alter the observed kinetic. A U. maydis triple mutant of cmu1, the isochorismatase coding gene um12021 and shy1 encoding a salicylate hydroxylase was reduced in virulence compared to any single or double mutants, suggesting an interplay of three U. maydis enzymes in suppressing SA pathway. By performing immunoprecipitation (IP) of Cmu1 from infected leave tissues followed by mass spectrometry, the maize protein Cmi1 (Cmu1 interactor 1) could be identified as an interactor. In vitro pull-down experiments confirmed the interaction between Cmu1 and Cmi1. Recombinant Cmi1 inhibited the chorismate mutase activity of Cmu1. The expression of cmi1 is strongly induced upon the infection of U. maydis, indicating that it is likely a pathogenesis related (PR) protein. Hydrogen-Deuterium exchange mass spectrometry (HDX/MS) mapped the interaction interface between Cmu1 and Cmi1, which involved the loop region of Cmu1. Truncation of the loop in Cmu1, which abolished the interaction of Cmu1 with Cmi1, showed only partial complementation of CL13Δcmu1 mutants, suggesting that the interaction between Cmu1 and Cmi1 may be relevant for the virulence of U. maydis.|