DataSheet_1_New Insights into rice pyrimidine catabolic enzymes.pdf

IntroductionRice 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.MethodsThe 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.ResultsThe 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.DiscussionOsDHP, 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.”

水稻作为全球主要的粮食来源，其产量受非生物胁迫的影响，这种胁迫由气候变化及其他因素所引发。近期，由二氢嘧啶还原代谢途径催化，该途径由二氢嘧啶脱氢酶（DHPD）、二氢嘧啶酶（DHP）和β-脒基丙酮酸酶（β-UP）等酶参与，已成为水稻非生物胁迫响应中的一个潜在参与者。
方法：水稻酶作为重组蛋白被生产，其中两种酶被进行了动力学特性表征。水稻二氢乳清酸脱氢酶（DHODH），一种常与DHPD混淆的嘧啶生物合成酶，也被进行了表征。对盐敏感和耐盐的水稻幼苗施加盐胁迫（24小时），并通过质谱法测定叶片中的代谢物。
结果：OsDHPD序列与哺乳动物DHPD的C端半部分同源，保留了FMN和尿嘧啶结合位点，但缺乏铁硫簇、FAD和NADPH的结合位点。OsDHPD，经过截短以消除叶绿体靶向肽段，是可溶的，但无活性。在数据库中搜索与哺乳动物DHPD N端半部分同源的、能够作为辅还原剂的肽段未成功。OsDHODH表现出与其他植物DHODHs相似的动力学参数。OsDHP，经过截短以去除信号序列，表现出kcat/Km = 3.6 x 10^3 s^-1M^-1。Osb-UP表现出kcat/Km = 1.8 x 10^4 s^-1M^-1。短期盐暴露对盐敏感和耐盐植物叶片中脒类中间体二氢尿嘧啶和脒基丙酮酸的水平影响不显著。尿酸胺，一种嘌呤代谢的脒类代谢物，在耐盐品种中显著高于敏感品种之一。
讨论：OsDHP，作为首次被表征的植物酶，表现出较低的动力学效率，但其活性可能受到截短的影响。Osb-UP表现出与次级代谢酶相似的动力学参数。两种途径代谢物的水平在敏感和耐盐品种中相似，并且似乎不受短期盐暴露的影响。