Characterization of the Pyrimidine Catabolic Pathway of Oryza sativa

Abstract

ObjectiveTo characterize plant pyrimidine catabolic enzymes and to identify potential candidate for additional enzymes participating in the pathwayRice is a primary global food source and is an important crop in Colombia. Its production is affected by abiotic stress, caused by climate change and other factors. Recently, genes of the pyrimidine catabolic pathway have emerged as potential participants in the abiotic stress response of plants.In mammals, three enzymes are involved in the degradation of uracil or thymine. The first is dihydropyrimidine dehydrogenase (DHPD), the second is dihydropyrimidinase (DHP) and the last is β‐ureidopropionase (β‐UP). Liu and coworkers (2009) found that overexpression of DHPD in rice plants (OsDHPD) results in tolerance to salt and drought stress, however, in that study OsDHPD was misidentified as a type I dihydroorotate dehydrogenase. DHP appears to be involved in cold stress‐response in grass (Xuan et al., 2013). In addition, the overexpression of β‐UP in transgenic tomatoes plants made them resistant to heat during anthesis (Shen et al., 2014). The role of pyrimidine catabolism in stress tolerance could be related to this pathway's production of precursors for proline biosynthesis. In plants, a complete characterization of the pathway is lacking. Comparison of plant and mammalian DHPD sequences showed that the plant protein lacks a domain needed for binding NADH(P), FAD, and Fe/S clusters, suggesting that another, unidentified, partner protein should be interacting with OsDHPD.ResultsWe cloned and expressed three enzymes of the pathway, OsDHPD, OsDHP and Osβ‐UP. The third enzyme, Osβ‐UP, was soluble, and preliminary kinetics revealed a Km 0.85 ± 0.11 mM for 3‐ureidopropionic acid, and a specific activity apparently 10‐fold faster (Vmax 384.3 ± 12.0 nmol/min/mg) than the Arabidopsis thaliana recombinant enzyme (30 nmol/min/mg) (Walsh et al., 2001). Truncations lacking chloroplast localization signals were required to produce soluble recombinant proteins of the first two enzymes. Truncated OsDHPD, was soluble, but inactive, presumably because it requires a partner protein for catalysis. By screening the rice database for proteins with the appropriate combination of cofactors we identified two potential candidates that may act as partners of OsDHPD; the first was a salt‐induced chloroplast ferredoxin, and the second was a rice chloroplast “fruit protein”. Both were cloned and expressed, and testing of interactions with OsDHPD on native gels are in progress. In addition, we produced polyclonal antibodies to OsDHPD to permit pull‐down assays of chloroplast extracts.ConclusionsThree rice pyrimidine catabolic enzymes were cloned, expressed and purified. Potential candidates for the missing protein in the pathway were identified using bioinformatics tools, and antibodies to OsDHPD were generated. Together these approaches may allow identification of the missing participant in pyrimidine catabolism in plants.Support or Funding InformationThe research was supported by funding from Colciencias project 120471250531, and from the Facultad de Ciencias and the Vicerrectoria de Investigaciones (Universidad de los Andes, Colombia).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.