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Appl. Environ. Microbiol. doi:10.1128/AEM.02569-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of Ustilago maydis allows target validation of DHODH inhibitors in vivo

Faculty of Medicine, Philipps-University Marburg, Karl-von-Frisch-Str. 1, D-35033 Marburg, Germany; Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany

^* To whom correspondence should be addressed. Email: boelker{at}staff.uni-marburg.de.

	Abstract

Dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We could show that pyr4 contains a functional mitochondrial targeting signal. Deletion of pyr4 resulted in auxotrophy for uracil, enhanced sensitivity to UV irradiation and loss of pathogenicity on corn plants. Biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q₆ and is resistant to several commonly used DHODH inhibitors. Here we show that expression of human DHODH fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild type cells were resistant to the DHODH inhibitor brequinar, strains expressing human DHODH became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used as sensitive in vivo assay for the development of novel drugs specifically targeted at either human or fungal DHODH.