Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis.
Caffeic acid 3-O-methyltransferase (COMT) catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, and is believed to occupy a pivotal position in the lignin biosynthetic pathway. A cDNA (TaCM) was identified from wheat and it was found to be expressed constitutively in stem, leaf and root tissues. The deduced amino acid sequence of TaCM showed a high degree of identity with COMT from other plants, particularly in SAM binding motif and the residues responsible for catalytic and substrate specificity. The predicted TaCM three-dimensional structure is very similar with a COMT from alfalfa (MsCOMT), and TaCM protein had high immunoreactive activity with MsCOMT antibody. Kinetic analysis indicated that the recombinant TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leads to S lignin via aldehyde precursors. Authority of TaCM encoding a COMT was confirmed by the expression of antisense TaCM gene in transgenic tobacco which specifically down-regulated the COMT enzyme activity. Lignin analysis showed that the reduction in COMT activity resulted in a marginal decrease in lignin content but sharp reduction in the syringl lignin. Furthermore, the TaCM protein exhibited a strong activity towards ester precursors including caffeoyl-CoA and 5-hydroxyferuloyl-CoA. Our results demonstrate that TaCM is a typical COMT involved in lignin biosynthesis. It also supports the notion, in agreement with a structural analysis, that COMT has a broad substrate preference.
Ma
Qing-Hu
QH
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing, China. mqh@ibcas.ac.cn
Xu
Yang
Y
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000378:metabolism; D017343:Genes, Plant; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis*; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000378:metabolism; D014908:Triticum / Q000201:enzymology*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D008031:Lignin; D008780:Methyltransferases; C019608:caffeate O-methyltransferase