CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.
We characterized a new cytochrome P450 monooxygenase (P450), CYP724B2, from tomato (Lycopersicon esculentum). CYP724B2 showed 42% and 62% amino acid sequence identity with Arabidopsis DWARF4/CYP90B1 and rice DWARF11/CYP724B1 respectively. Functional assay of CYP724B2 heterologously expressed in insect cells revealed that CYP724B2 catalyzes C-22 hydroxylation of campesterol, indicating that CYP724B2 is a C-22 hydroxylase. We also isolated a tomato CYP90B homolog (CYP90B3) and found that CYP90B3 is a C-22 hydroxylase as well. CYP724B2 and CYP90B3 showed substrate specificities similar to each other toward the biosynthetic intermediate compounds from campesterol to campestanol. Campesterol was the best substrate, and (24R)-ergost-4-en-3-one was also metabolized to the C-22 hydroxylated product to some extent. On the other hand, the P450s catalyzed C-22 hydroxylation of (24R)-5alpha-ergostan-3-one and campestanol at a trace level, indicating that the compounds after C-5alpha reduction are poor substrates of CYP724B2 and CYP90B3. In addition, cholesterol (C27 sterol) and sitosterol (C29 sterol) were also converted to C-22 hydroxylated products by the P450s. Furthermore, CYP724B2 and CYP90B3 genes were ubiquitously expressed, and their transcript levels were down-regulated by the exogenous application of brassinolide. These findings strongly suggest that CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.
Ohnishi
Toshiyuki
T
Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan.
Watanabe
Bunta
B
Sakata
Kanzo
K
Mizutani
Masaharu
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D020224:Expressed Sequence Tags / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010840:Phytosterols / Q000096:biosynthesis* / Q000378:metabolism; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; D010840:Phytosterols; D018749:RNA, Plant; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System