New Insights into rice pyrimidine catabolic enzymes

Abstract

Introduction: Rice is a primary global food source, and its production is affected by abiotic stress, caused by climate change and other factors. Recently, the pyrimidine reductive catabolic pathway, catalyzed by dihydropyrimidine dehydrogenase (DHPD), dihydropyrimidinase (DHP) and β-ureidopropionase (β-UP), has emerged as a potential participant in the abiotic stress response of rice.

Methods: The rice enzymes were produced as recombinant proteins, and two were kinetically characterized. Rice dihydroorotate dehydrogenase (DHODH), an enzyme of pyrimidine biosynthesis often confused with DHPD, was also characterized. Salt-sensitive and salt-resistant rice seedlings were subjected to salt stress (24 h) and metabolites in leaves were determined by mass spectrometry.

Results: The OsDHPD sequence was homologous to the C-terminal half of mammalian DHPD, conserving FMN and uracil binding sites, but lacked sites for Fe/S clusters, FAD, and NADPH. OsDHPD, truncated to eliminate the chloroplast targeting peptide, was soluble, but inactive. Database searches for polypeptides homologous to the N-terminal half of mammalian DHPD, that could act as co-reductants, were unsuccessful. OsDHODH exhibited kinetic parameters similar to those of other plant DHODHs. OsDHP, truncated to remove a signal sequence, exhibited a kcat/Km = 3.6 x 103 s-1M-1. Osb-UP exhibited a kcat/Km = 1.8 x 104 s-1M-1. Short-term salt exposure caused insignificant differences in the levels of the ureide intermediates dihydrouracil and ureidopropionate in leaves of salt-sensitive and salt-resistant plants. Allantoin, a ureide metabolite of purine catabolism, was found to be significantly higher in the resistant cultivar compared to one of the sensitive cultivars.

Discussion: OsDHP, the first plant enzyme to be characterized, showed low kinetic efficiency, but its activity may have been affected by truncation. Osb-UP exhibited kinetic parameters in the range of enzymes of secondary metabolism. Levels of two pathway metabolites were similar in sensitive and resistant cultivars and appeared to be unaffected by short-term salt exposure."

Keywords: Oryza sativa; abiotic stress; dihydroorotate dehydrogenase; dihydropyrimidinase; dihydropyrimidine dehydrogenase; plants; pyrimidine catabolism; ßureidopropionase.

Figure 1

A schematic alignment of the mammalian DHPD and OsDHPD. The upper bar represents SsDHPD (Genbank U09179.2), and shows approximate locations of residues involved in binding cofactors and substrate, that are identified in the crystal structure (Dobritzsch et al., 2001). Position of residues interacting with FMN are indicated with vertical blue lines, positions of residues interacting with substrate are shown with vertical red lines, and the positions of four iron sulfur cluster are shown in yellow. The positions of residues involved in FAD and NADPH binding are indicated by the vertical black lines above the bar. The lower bar represents OsDHPD, and the region of homology with SsDHPD is highlighted in gray. The conserved FMN and uracil binding residues are shown below the bar. Regions that are not homologous are shown in white and green, the latter indicating the chloroplast targeting sequence.

Figure 2

Steady state kinetics of purified recombinant OsDHODH. (A) Saturation curve for L-dihydroorotate. (B) Saturation curve for decylubiqinone, Q_d.

Figure 3

Steady state kinetics of purified recombinant OsDHP-T1. Saturation curve for dihydrouracil. Data were fitted to the Michaelis-Menten equation, v = (V_max · [S])/(K_m + [S]).

Figure 4

Steady state kinetics of purified recombinant Osß-UP. (A), Saturation curve for ß-ureidopropionate. (B), Saturation curve for ß-ureidoisobutyric acid. Data were fitted to the Michaelis-Menten equation, v = (V_max · [S])/(K_m + [S]).

Figure 5

Levels of selected metabolites of pyrimidine catabolism in rice leaves during 24 hours of exposure to salt stress in salt-sensitive and salt-tolerant cultivars. Three rice cultivars were grown hydroponically in a phytotron, and 200 mM NaCl was added to growth media on day 17. Koshihikari and Azucena are salt-sensitive, and IR64 is salt-tolerant. Leaves were harvested during a 24-hour period post-treatment, and the levels of selected metabolites of pyrimidine catabolism, uracil (A), dihydrouracil (B), and ureidopropionic acid (C), were measured. Values on the y-axis represent the integrated area of peaks associated with the metabolite of interest, normalized to mg protein in the sample. It is important to note that the levels of different compounds cannot be compared. No significant differences in metabolite levels were observed in a two-way ANOVA using GraphPad Prism 9.

Figure 6

Levels of selected metabolites of purine catabolism in rice leaves during 24 hours of exposure to salt stress. Three rice cultivars were grown hydroponically in a phytotron, and 200 mM NaCl was added to growth media on day 17. Koshihikari and Azucena are salt-sensitive, and IR64 is salt-tolerant. Leaves were harvested during a 24-hour period post-treatment, and the levels of metabolites were measured. Allantoin (B) and allantoic acid (A) are the products of the fourth and fifth steps, respectively, of xanthine degradation. Values on the y-axis represent the integrated area of peaks associated with the metabolite of interest, normalized to mg protein in the sample. It is important to note that the levels of different compounds cannot be compared. Data were analyzed in a two-way ANOVA using GraphPad Prism 9 (*P values 0.01 - 0.05, ** P values 0.001 – 0.01, *** P values 0.0001 – 0.001).