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

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 kcat app of 30 ± 1 s-1, and a Km app of 20 ± 1 µM for L-dihydroorotate, and a Km app= 30 ± 3 µM for decylubiquinone (Qd). PiDHODH exhibited an apparent specific activity of 104 ± 1 µmol min-1 mg-1, a kcat app of 75 ± 1 s-1, and a Km app of 57 ± 3 µM for L-dihydroorotate, and a Km 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 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.

Pyrimidine Metabolism: Dynamic and Versatile Pathways in Pathogens and Cellular Development.

The importance of pyrimidines lies in the fact that they are structural components of a broad spectrum of key molecules that participate in diverse cellular functions, such as synthesis of DNA, RNA, lipids, and carbohydrates. Pyrimidine metabolism encompasses all enzymes involved in the synthesis, degradation, salvage, interconversion and transport of these molecules. In this review, we summarize recent publications that document how pyrimidine metabolism changes under a variety of conditions, including, when possible, those studies based on techniques of genomics, transcriptomics, proteomics, and metabolomics. First, we briefly look at the dynamics of pyrimidine metabolism during nonpathogenic cellular events. We then focus on changes that pathogen infections cause in the pyrimidine metabolism of their host. Next, we discuss the effects of antimetabolites and inhibitors, and finally we consider the consequences of genetic manipulations, such as knock-downs, knock-outs, and knock-ins, of pyrimidine enzymes on pyrimidine metabolism in the cell.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.