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 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.

FIG. 1.

U. maydis DHODH is targeted to the mitochondria. The mitochondrial targeting sequence of U. maydis DHODH was fused to GFP and expressed in haploid U. maydis cells. (A) The colocalization of the U. maydis DHODH fused to amino acids 1 to 73 of GFP (DHODH_1-73GFP) with mitochondria stained by Mitotracker rhodamine B indicates the targeting of U. maydis DHODH to the mitochondria. DIC, differential interference contrast. (B) Schematic drawing of different GFP constructs used for the localization of U. maydis DHODH (UmDHODH). The mitochondrial targeting sequences (mTS) and the transmembrane domains (TM) are indicated. (C) Confocal fluorescence micrographs of FM4-64-stained cells expressing DHODH fused to amino acids 1 to 110 of GFP (DHODH_1-110GFP) or DHODH_1-73GFP reveal no differences in localization.

FIG. 2.

Expression of recombinant U. maydis dihydroorotate dehydrogenase in E. coli and Western analysis of mitochondrial proteins. (A) The N-terminally truncated U. maydis DHODH (UmDHODH-ΔN) was expressed in E. coli and purified by affinity chromatography. Shown is a Coomassie-stained SDS gel of the purified protein (2 μg). (B) Western blot of purified U. maydis DHODH-ΔN. The purified protein (1 μg) was separated by SDS-PAGE and immunostained with rabbit polyclonal anti-human DHODH serum. (C) Mitochondria were prepared from an FB1 Δpyr4 mutant strain transformed with U. maydis DHODH (UmDHODH), the wild-type (WT) FB1 strain, an FB1 Δpyr4 mutant strain (pyr4−), and an FB1 Δpyr4 mutant strain expressing the chimeric U. maydis-human DHODH construct (Um-HsDHODH); separated by SDS-PAGE; and immunostained with rabbit polyclonal anti-human DHODH serum.

FIG. 3.

The functional expression of human dihydroorotate dehydrogenase in U. maydis complements the growth defect of Δpyr4 mutants. (A) Full-length and chimeric DHODH genes were expressed under the control of the U. maydis pyr4 (DHODH) promoter (P_pyr4). Dark gray, U. maydis DHODH (UmDHODH) open reading frame; light gray, human DHODH (HsDHODH) open reading frame; mTS, mitochondrial targeting sequence; TM, transmembrane domain; Um-HsDHODH, chimeric U. maydis-human DHODH construct. (B) Wild-type (WT) strain FB1, the FB1 Δpyr4 mutant (FB1Δpyr4), and transformed strains were grown on full medium (yeast extract-peptone-dextrose [YEPD]) and minimal medium without uracil [MM (−Uracil)].

FIG. 4.

The depletion of DHODH results in enhanced sensitivity to UV radiation. (A) Cells expressing the pyr4 open reading frame under the control of the arabinose-inducible crg promoter (Pcrg::pyr4) are highly sensitive to UV radiation if the expression of pyr4 is repressed in glucose-containing medium. If the medium contains arabinose, no difference from wild-type (WT) cells can be observed. (B) The UV radiation sensitivity of conditional pyr4 mutants (Pcrg::pyr4) grown under repressing conditions (in the presence of glucose) is alleviated if uridine/uracil, cytidine, or orotate is added to the medium. DHO is unable to restore the radiation resistance.

FIG. 5.

U. maydis strains expressing human DHODH are inhibited by brequinar. U. maydis wild-type cells (WT), pyr4 mutants (pyr4), and pyr4 mutants transformed with either full-length U. maydis DHODH (pyr4/UmDHODH) or the chimeric U. maydis-human DHODH (pyr4/Um-HsDHODH) were grown in minimal medium. Cell growth was determined 16 h after the addition of the inhibitor brequinar (10 μM).