Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism.
Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.
Ohnishi
Toshiyuki
T
Institute for Chemical Research, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan.
Nomura
Takahito
T
Watanabe
Bunta
B
Ohta
Daisaku
D
Yokota
Takao
T
Miyagawa
Hisashi
H
Sakata
Kanzo
K
Mizutani
Masaharu
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010802:Phylogeny; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013260:Steroids, Heterocyclic / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D013260:Steroids, Heterocyclic; D003577:Cytochrome P-450 Enzyme System