Abstract
The oomycete Phytophthora infestans is the causal agent of tomato and potato late blight, a disease that causes tremendous economic losses in the production of solanaceous crops. The similarities between oomycetes and the apicomplexa led us to hypothesize that dihydroorotate dehydrogenase (DHODH), the enzyme catalyzing the fourth step in pyrimidine biosynthetic pathway, and a validated drug target in treatment of malaria, could be a potential target for controlling P. infestans growth. In eukaryotes, class 2 DHODHs are mitochondrially associated ubiquinone-linked enzymes that catalyze the fourth, and only redox step of de novo pyrimidine biosynthesis. We characterized the enzymes from both the pathogen and a host, Solanum tuberosum. Plant DHODHs are known to be class 2 enzymes. Sequence analysis suggested that the pathogen enzyme (PiDHODHs) also belongs to this class. We confirmed the mitochondrial localization of GFP-PiDHODH showing colocalization with mCherry-labeled ATPase in a transgenic pathogen. N-terminally truncated versions of the two DHODHs were overproduced in E. coli, purified, and kinetically characterized. StDHODH exhibited a apparent specific activity of 41 ± 1 μmol min-1 mg-1, a kcatapp of 30 ± 1 s-1, and a Kmapp of 20 ± 1 μM for L-dihydroorotate, and a Kmapp= 30 ± 3 μM for decylubiquinone (Qd). PiDHODH exhibited an apparent specific activity of 104 ± 1 μmol min-1 mg-1, a kcatapp of 75 ± 1 s-1, and a Kmapp of 57 ± 3 μM for L-dihydroorotate, and a Kmapp of 15 ± 1 μM for Qd. The two enzymes exhibited different activities with different quinones and napthoquinone derivatives, and different sensitivities to compounds known to cause inhibition of DHODHs from other organisms. The IC50 for A77 1726, a nanomolar inhibitor of human DHODH, was 2.9 ± 0.6 mM for StDHODH, and 79 ± 1 μM for PiDHODH. In vivo, 0.5 mM A77 1726 decreased mycelial growth by approximately 50%, after 92 h. Collectively, our findings suggest that the PiDHODH could be a target for selective inhibitors and we provide a biochemical background for the development of compounds that could be helpful for the control of the pathogen, opening the way to protein crystallization.
Highlights
Within the Phylum Oomycota, the species of the genus Phytophthora are all considered devastating pathogens of crops and landscape plants, and are responsible annually for huge economic losses worldwide (Lamour et al, 2007; Attard et al, 2008)
The derived amino acid sequence contained typical features of the class 2 DHODHs, including an N-terminal extension, a transmembrane domain, a conserved serine (S277) that aligned with the catalytic serines of E. coli (S175), human (S215), and P. falciparum (S345) DHODHs, and conserved residues for binding the electron acceptor, flavin mononucleotide (FMN), and orotate (Figure 1)
The identity with other plant DHODHs was high (Zea mays 73%, A. thaliana 77%), and revealed that the enzyme was more similar to the class 2 DHODHs (Homo sapiens 49%, P. falciparum 30%, P. infestans 48%, E. coli 41%) (Supplementary Table 2) than to class 1 DHODHs (T. cruzi 21%, S. cerevisiae 21%)
Summary
Within the Phylum Oomycota, the species of the genus Phytophthora are all considered devastating pathogens of crops and landscape plants, and are responsible annually for huge economic losses worldwide (Lamour et al, 2007; Attard et al, 2008). Phytophthora infestans causes late blight disease in potato, tomato, and other solanaceous crops. In these hosts, the entire plant is destroyed within a few days after the first lesions are observed (Fry, 2008). In most eukaryotes, including plants (Witz et al, 2012) and some plant pathogenic fungi (Zameitat et al, 2007), class 2 DHODHs are associated with the outer surface of the inner mitochondrial membrane (Rawls et al, 2000) and transfer electrons from dihydroorotate oxidation to ubiquinone in the respiratory chain. Class 2 DHODHs are found in gramnegative bacteria (Björnberg et al, 1999), anchored to the inner side of the periplasmic side of the inner cytoplasmic membrane, transferring electrons to the respiratory chain
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