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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 k cat  app  of 30 ± 1 s -1 , and a K m  app  of 20 ± 1 μM for  L -dihydroorotate, and a K m  app = 30 ± 3 μM for decylubiquinone (Qd). PiDHODH exhibited an apparent specific activity of 104 ± 1 μmol min -1  mg -1 , a k cat  app  of 75 ± 1 s -1 , and a K m  app  of 57 ± 3 μM for  L -dihydroorotate, and a K m  app  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 IC 50  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  Pi DHODH 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.

Phytophthora infestans Dihydroorotate Dehydrogenase Is a Potential Target for Chemical Control – A Comparison With the Enzyme From Solanum tuberosum