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1.  Abscinazole-E3M, a practical inhibitor of abscisic acid 8′-hydroxylase for improving drought tolerance 
Scientific Reports  2016;6:37060.
Abscisic acid (ABA) is an essential phytohormone that regulates plant water use and drought tolerance. However, agricultural applications of ABA have been limited because of its rapid inactivation in plants, which involves hydroxylation of ABA by ABA 8′-hydroxylase (CYP707A). We previously developed a selective inhibitor of CYP707A, (−)-Abz-E2B, by structurally modifying S-uniconazole, which functions as an inhibitor of CYP707A and as a gibberellin biosynthetic enzyme. However, its synthetic yield is too low for practical applications. Therefore, we designed novel CYP707A inhibitors, Abz-T compounds, that have simpler structures in which the 1,2,3-triazolyl ring of (−)-Abz-E2B has been replaced with a triple bond. They were successfully synthesised in shorter steps, resulting in greater yields than that of (−)-Abz-E2B. In the enzymatic assays, one of the Abz-T compounds, (−)-Abz-E3M, acted as a strong and selective inhibitor of CYP707A, similar to (−)-Abz-E2B. Analysis of the biological effects in Arabidopsis revealed that (−)-Abz-E3M enhanced ABA’s effects more than (−)-Abz-E2B in seed germination and in the expression of ABA-responsive genes. Treatment with (−)-Abz-E3M induced stomatal closure and improved drought tolerance in Arabidopsis. Furthermore, (−)-Abz-E3M also increased the ABA response in rice and maize. Thus, (−)-Abz-E3M is a more practical and effective inhibitor of CYP707A than (−)-Abz-E2B.
PMCID: PMC5107945  PMID: 27841331
2.  Sustained low abscisic acid levels increase seedling vigor under cold stress in rice (Oryza sativa L.) 
Scientific Reports  2015;5:13819.
Stress-induced abscisic acid (ABA) is mainly catabolized by ABA 8′-hydroxylase (ABA8ox), which also strictly regulates endogenous ABA levels. Although three members of the ABA8ox gene family are conserved in rice, it is not clear which stressors induce expression of these genes. Here, we found that OsABA8ox1 was induced by cold stress within 24 h and that OsABA8ox2 and OsABA8ox3 were not. In contrast, OsABA8ox2 and OsABA8ox3 were ABA-inducible, but OsABA8ox1 was not. OsABA8ox1, OsABA8ox2, and OsABA8ox3 restored germination of a cyp707a1/a2/a3 triple mutant of Arabidopsis to rates comparable to those of the wild type, indicating that OsABA8ox1, OsABA8ox2, and OsABA8ox3 function as ABA-catabolic genes in vivo. Transgenic rice lines overexpressing OsABA8ox1 showed decreased levels of ABA and increased seedling vigor at 15 °C. These results indicate that sustained low levels of ABA lead to increased seedling vigor during cold stress. On the other hand, excessively low endogenous ABA levels caused reduced drought and cold tolerance, although some of the transgenic rice lines expressing OsABA8ox1 at moderate levels did not show these harmful effects. Adequate regulation of endogenous ABA levels is thought to be crucial for maintaining seedling vigor under cold stress and for cold and drought tolerance in rice.
PMCID: PMC4563555  PMID: 26350634
3.  Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8 
Structures of hypoxanthine-guanine phosphoribosyltransferase from T. thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP are reported at 2.1, 1.9 and 2.2 Å resolution, respectively.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of IMP or GMP from α-d-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. Structures of HGPRTase from Thermus thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP have been determined at 2.1, 1.9 and 2.2 Å resolution, respectively. The overall fold of the IMP complex was similar to that of the unliganded form, but the main-chain and side-chain atoms of the active site moved to accommodate IMP. The overall folds of the IMP and GMP complexes were almost identical to each other. Structural comparison of the T. thermo­philus HB8 enzyme with 6-­oxopurine PRTases for which structures have been determined showed that these enzymes can be tentatively divided into groups I and II and that the T. thermophilus HB8 enzyme belongs to group I. The group II enzymes are characterized by an N-­terminal extension with additional secondary elements and a long loop connecting the second α-helix and β-strand compared with the group I enzymes.
PMCID: PMC2917284  PMID: 20693661
transferases; Rossmann fold; purine nucleotide biosynthetic pathway
4.  Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin 
Invertebrate Neuroscience   2008;8(2):71-81.
Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.
PMCID: PMC2413115  PMID: 18338186
Acetylcholine binding protein (Lymnaea stagnalis); Crystal structures; Neonicotinoids; Nicotinic acetylcholine receptors; Ion channels

Results 1-4 (4)