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

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 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 Q 6 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.

De NovoPyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans

“…1), which include the enzymes largely from mammals, higher eukaryotes, and prokaryotes as well (10, 12, 18–20). Family 2 DHODH enzymes from mammals and higher eukaryotes typically possess N-terminal extensions that facilitate their mitochondrial localization and association with mitochondrial membrane.…”

“…Studies from humans, Plasmodium falciparum, and other fungal species, including Candida albicans , Schizosaccharomyces pombe, and Ustilago maydis, demonstrate the structure of DHODH enzymes to be comprised of two domains, an N-terminal alpha-helical domain and a large C-terminal domain connected by an extended loop (9, 15, 18, 22). The predicted annotation of the C. neoformans var.…”

“…C. neoformans is insensitive to brequinar, an inhibitor of the human DHODH (data not shown). This is unsurprising, as the closely related basidiomycete Ustilago maydis , which encodes a family 2 DHODH with the conserved N-terminal extension, is also resistant to brequinar (18). The resistance in these fungi is likely not due to permeability of the drug, as replacement of the U. maydis DHODH with the human enzyme results in brequinar sensitivity.…”

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

“…The enzyme dihydroorotate dihydrogenase (DHOase-URA1) converts dihydroooratate into orotate, and DHOase inhibitors have been successfully tested as antiproliferative agents in neoplastic growth, as suppressors of immunological responses (Chen et al 1992; Greene et al 1995; Liu et al, 2000, Khutornenko et al, 2010), and also as inhibitors of the growth of Toxoplasma gondii (Hegewald et al 2013). In the basidiomycete Ustilago maydis, the pyr4 gene encodes dihydroorotate dehydrogenase, and its absence results in loss of pathogenicity and uracil auxotrophy (Banuett, 1995; Bölker, 2001; Zameitat, et al, 2007). Previous work by Morrow et al (2012) showed that de novo synthesis of purine derivatives such as GTP is also important for virulence in C. neoformans .…”

Dihydroorotate dehydrogenase fromSaccharomyces cerevisiae: spectroscopic investigations with the recombinant enzyme throw light on catalytic properties and metabolism of fumarate analogues

“…Recently, the recombinant enzyme was obtained after heterologous expression in Escherichia coli (Zameitat et al ., 2004). The newest approach in dihydroorotate dehydrogenase research is the development of transgenic organisms for the use of target validation (Painter et al ., 2007; Zameitat et al ., 2007) and underlines the important role of the enzyme.…”