Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica.
In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-L-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.
Yoshihara
Noriko
N
The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan.
Fukuchi-Mizutani
Masako
M
Okuhara
Hiroaki
H
Tanaka
Yoshikazu
Y
Yabuya
Tsutomu
T
D000595:Amino Acid Sequence; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D035264:Flowers / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D031323:Iris Plant / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011497:Protein O-Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D012333:RNA, Messenger; D011994:Recombinant Proteins; D012435:S-Adenosylhomocysteine; D011497:Protein O-Methyltransferase