Characterization of three cinnamyl alcohol dehydrogenases from Carthamus tinctorius
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概要
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We report the isolation and characterization of three cDNAs encoding cinnamyl alcohol dehydrogenase (CAD) from Carthamus tinctorius (safflower). All three recombinant CADs were able to reduce coniferaldehyde and sinapaldehyde into coniferyl alcohol and sinapyl alcohol, respectively, and were designated as CtCAD1, CtCAD2, and CtCAD3. Phylogenetic analysis of CAD amino acid sequences and homology modeling revealed that CtCAD1 and CtCAD3 were closely related to the sinapaldehyde-specific aspen (Populus tremuloides) sinapyl alcohol dehydrogenase (PtreSAD). CtCAD2 was in a clade containing class I plant CADs. Gas chromatography-mass spectrometry-based kinetic analysis using two different substrates, coniferaldehyde and sinapaldehyde, indicated that the CtCADs showed no strong preference for either substrate. CtCAD2 has the highest catalytic efficiency (kcat/Km) (81.49 mM−1 min−1 and 95.3 mM−1 min−1 for coniferaldehyde and sinapaldehyde, respectively) compared with the other CtCADs. Inhibition kinetics showed that coniferaldehyde was a stronger inhibitor than sinapaldehyde for all CtCADs. Quantitative real-time PCR revealed that CtCAD2 was expressed at higher levels than CtCAD1 and CtCAD3 in all samples, except developing seeds at 3 days after flowering, where CtCAD1 had a higher expression level. In plant protein assays with coniferaldehyde and sinapaldehyde, plant protein extracted from seeds at 7 days after flowering, showed the highest specific activity. The product yields in plant protein assays were strongly correlated with gene expressions of CtCAD2 and CtCAD3 in the respective organs.
著者
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Nakatsubo Tomoyuki
Research Institute For Sustainable Humanosphere Kyoto University
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Umezawa Toshiaki
Research Institute For Sustainable Humanosphere Kyoto University
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Suzuki Shiro
Research Institute For Sustainable Humanosphere Kyoto University
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HATTORI Takefumi
Research Institute for Sustainable Humanosphere, Kyoto University
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Ragamustari Safendrri
Research Institute for Sustainable Humanosphere, Kyoto University
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Shiraiwa Naoko
Research Institute for Sustainable Humanosphere, Kyoto University
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