Shigeki Saito

Kyoto University, Kyoto, Kyoto-fu, Japan

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Publications (6)26.41 Total impact

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    ABSTRACT: Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8'-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K(i)=8.0 nM) of ABA 8'-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA(4) did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8'-hydroxylase inhibitory activity.
    Bioscience Biotechnology and Biochemistry 08/2006; 70(7):1731-9. · 1.21 Impact Factor
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    ABSTRACT: Delta22-unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with beta-sitosterol to produce stigmasterol (CYP710A1, K(m) = 1.0 microM and kinetic constant [k(cat)] = 0.53 min(-1); CYP710A11, K(m) = 3.7 microM and k(cat) = 10 min(-1)). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6- to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and beta-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:beta-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the Delta(22)-unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.
    The Plant Cell 05/2006; 18(4):1008-22. · 9.58 Impact Factor
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    ABSTRACT: (1'S*,2'S*)-(+/-)-6-Nor-2',3'-dihydro-4'-deoxo-ABA (2) was designed and synthesized as a candidate lead compound for developing a potent and specific inhibitor of ABA 8'-hydroxylase. This compound acted as an effective competitive inhibitor of the enzyme, with a K(I) value of 0.40microM, without exhibiting ABA activity. However, compound 2 also functioned as an enzyme substrate, making it a short-lived inhibitor. The 8'-difluorinated derivative of 2 (4) was synthesized as a long-lasting alternative. Compound 4 resisted 8'-hydroxylation, but inhibited ABA 8'-hydroxylation as effectively as 2. These results suggest that compound 2 is a useful lead compound for the future design and development of an ideal ABA 8'-hydroxylase inhibitor.
    Bioorganic & Medicinal Chemistry Letters 02/2006; 15(23):5226-9. · 2.33 Impact Factor
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    ABSTRACT: Plant hormone abscisic acid (ABA) is an important factor for conferring drought stress resistance on plants. Therefore, small molecules that regulate ABA levels in plants can be useful both for investigating functions of ABA and for developing new plant growth regulators. Abscisic acid (ABA) catabolism in plants is primarily regulated by ABA 8'-hydroxylase, which is a cytochrome P450 (P450). We tested known P450 inhibitors containing a triazole group and found that uniconazole-P inhibited ABA catabolism in cultured tobacco Bright Yellow-2 cells. In a structure-activity study of uniconazole, we found a more effective ABA catabolic inhibitor (diniconazole) than uniconazole-P. Diniconazole, a fungicide, acted as a potent competitive inhibitor of recombinant Arabidopsis ABA 8'-hydroxylase, CYP707A3, in an in vitro assay. Diniconazole-treated plants retained a higher ABA content and higher transcription levels of ABA response genes during rehydration than did untreated plants and were more drought stress tolerant than untreated plants. These results strongly suggest that ABA catabolic inhibitors that target ABA 8'-hydroxylase can regulate the ABA content of plants and conferred drought stress resistance on plants. The optical resolution of diniconazole revealed that the S-form isomer, which is a weak fungicidal isomer, was more active as an ABA catabolic inhibitor than was the R-form isomer.
    Bioorganic & Medicinal Chemistry 08/2005; 13(14):4491-8. · 2.95 Impact Factor
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    ABSTRACT: A major catabolic enzyme of the plant hormone abscisic acid (ABA) is the cytochrome P450 monooxygenase ABA 8'-hydroxylase. For designing a specific inhibitor of this enzyme, the substrate specificity and inhibition of CYP707A3, an ABA 8'-hydroxylase from Arabidopsis thaliana, was investigated using 45 structural analogues of ABA and compared to the structural requirements for ABA activity. Substrate recognition by the enzyme strictly required the 6'-methyl groups (C-8' and C-9'), which were unnecessary for ABA activity, whereas elimination of the 3-methyl (C-6) and 1'-hydroxyl groups, which significantly affected ABA activity, had little effect on the ability of analogues to competitively inhibit the enzyme. Fluorination at C-8' and C-9' resulted in resistance to 8'-hydroxylation and competitive inhibition of the enzyme. In particular, 8',8'-difluoro-ABA and 9',9'-difluoro-ABA yielded no enzyme reaction products and strongly inhibited the enzyme (K(I) = 0.16 and 0.25 microM, respectively).
    Bioorganic & Medicinal Chemistry 06/2005; 13(10):3359-70. · 2.95 Impact Factor
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    ABSTRACT: Abscisic acid (ABA) is involved in a number of critical processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, a physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. The hydroxylation at the 8'-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 8'-hydroxylation of (+)-ABA. First, all four CYP707A cDNAs were cloned from Arabidopsis and used for the production of the recombinant proteins in insect cells using a baculovirus system. The insect cells expressing CYP707A3 efficiently metabolized (+)-ABA to yield phaseic acid, the isomerized form of 8'-hydroxy-ABA. The microsomes from the insect cells exhibited very strong activity of 8'-hydroxylation of (+)-ABA (K(m) = 1.3 microm and k(cat) = 15 min(-1)). The solubilized CYP707A3 protein bound (+)-ABA with the binding constant K(s) = 3.5 microm, but did not bind (-)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 8'-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 8'-hydroxylase. The transcripts of the CYP707A genes increased in response to salt, osmotic, and dehydration stresses as well as ABA. These results establish that the CYP707A family plays a key role in regulating the ABA level through the 8'-hydroxylation of (+)-ABA.
    Plant physiology 05/2004; 134(4):1439-49. · 7.39 Impact Factor