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

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 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.

Kinetic Study of the Avocado Sunblotch Viroid Self-Cleavage Reaction Reveals Compensatory Effects between High-Pressure and High-Temperature: Implications for Origins of Life on Earth

“…Such conditions are also prevalent in terrestrial subsurface environments, which nurture a massive microbial reservoir, estimated at about 70% of the Earth’s microbial life [ 19 , 20 ]. Some of these bacteria, for instance the subseafloor Petrocella atlantisensis , are able to strive in the laboratory, if cultured under high hydrostatic pressure [ 21 ]. It is also imaginable that such extreme conditions of high-pressure high-temperature are ordinary on other rocky planets, or even exoplanets.…”

New Insights into rice pyrimidine catabolic enzymes

“…It is important to note that Class 2 DHODHs from eukaryotic organisms are usually produced as truncated recombinant proteins ( Hortua Triana et al., 2012 ), and the lack of these N-terminal sequences appear to have little effect on the kinetic parameters of the recombinant enzymes ( Ullrich et al., 2002 ). We purified OsDHODH, and compared its kinetic parameters to those of other class 2 plant biosynthetic DHODHs ( Ullrich et al., 2002 ; Garavito et al., 2019 ) ( Figure 2 , Table 2 ). The apparent K m s for dihydroorotate and the specific activities of the OsDHODH and Solanum tuberosum DHODH ( Garavito et al., 2019 ) are quite similar, however the apparent K m for decylubiquinone (Q d ) is 6-fold lower in the rice enzyme.…”

“…We purified OsDHODH, and compared its kinetic parameters to those of other class 2 plant biosynthetic DHODHs ( Ullrich et al., 2002 ; Garavito et al., 2019 ) ( Figure 2 , Table 2 ). The apparent K m s for dihydroorotate and the specific activities of the OsDHODH and Solanum tuberosum DHODH ( Garavito et al., 2019 ) are quite similar, however the apparent K m for decylubiquinone (Q d ) is 6-fold lower in the rice enzyme. Both plant enzymes exhibit 5-fold lower specific activities compared to the human enzyme.…”

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses

“…The compound NSC629301 selectively targeted to the damage checkpoint and repair (DDCR) genes in yeast, Saccharomyces cerevisiae ( Tamble et al, 2011 ). Recently, NSC71097 has been shown to have the effects on either inhibition or increase enzymatic activity of dihydroorotate dehydrogenase (DHODH) from a pathogenic plant fungus, oomycete, suggesting that NSC71097 acted as electron acceptors for DHODH of Phytophthora infestans ( Garavito et al, 2019 ). Thus far, there is neither a report on their antiviral screening nor antimicrobial activities.…”