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Applied and Environmental Microbiology, May 2007, p. 3371-3379, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02569-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo{triangledown}

Elke Zameitat,1 Gerald Freymark,2,{dagger} Cornelia D. Dietz,1 Monika Löffler,1 and Michael Bölker2*

Faculty of Medicine, Philipps University Marburg, Karl-von-Frisch-Str. 1, D-35033 Marburg, Germany,1 Department of Biology, Philipps University Marburg, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany2

Received 3 November 2006/ Accepted 7 March 2007

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 the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The 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 the expression of the human DHODH gene 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 the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.

* Corresponding author. Mailing address: Dept. of Biology, Philipps University Marburg, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany. Phone: 49-6421-2821536. Fax: 49-6421-2828971. E-mail: boelker{at}staff.uni-marburg.de

{triangledown} Published ahead of print on 16 March 2007.

{dagger} Present address: Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, Germany.

Applied and Environmental Microbiology, May 2007, p. 3371-3379, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02569-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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