cDNA cloning and characterization of a 3?/5?-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum

Abstract

Enzymatic O-methylation of plant secondary metabolites is an important mechanism for the inactivation of reactive hydroxyl groups and for the modification of their solubility. A cDNA clone (pFOMT3') encoding the gene for the 3'/5'-O-methylation of partially methylated flavonols was isolated from Chrysosplenium americanum (Saxifragaceae). We used a PCR fragment obtained with degenerate oligonucleotides designed from conserved regions of various O-methyltransferases (OMTs). The pFOMT3' cDNA sequence shows about 67-85% similarity to other plant OMT sequences. The recombinant protein expresses strict specificity for positions 3'/5' (meta) of partially methylated flavonols, but does not accept quercetin or caffeic acid for further methylation. Southern blot analysis of the genomic DNA probed with an OMT sequence suggests the presence of a number of related genes in this species, consistent with the multiple enzymatic methylations involved in the biosynthesis of polymethylated flavonols in this plant.

Evolution of Novel O-methyltransferases from the Vanilla planifolia Caffeic Acid O-methyltransferase

“…The similar C. americanum FOMT is 83% identical to the C. americanum COMT but was inactive with COMT substrates. The C. americanum FOMT was active on several flavonoids, but not myricetin (Gauthier et al, 1996).…”

“…All branches were supported by greater than 50% bootstrap percentages based on 1000 replications. GenBank accession numbers for the corresponding DNA sequences are: Allium cepa, CF442066 (Kuhl et al, 2004); Catharanthus roseus, AY028439 (Schroder et al, 2002); Chrysosplenium americanum, U16793 (Gauthier et al, 1998); Chrysosplenium americanum FOMT, U16794, (Gauthier et al, 1996); Clarkia breweri, AF006009 (Wang and Pichersky, 1997); Clarkia breweri IEMT, U86760 ; Coffea canephora, AF454631 (unpublished data); Eucalyptus gunnii, X74814 (Poeydomenge et al, 1994); Festuca arundinacea, AF153825 (unpublished data); Iris hollandica, AB183825 (unpublished data); Lolium perenne, AF010291 (McAlister et al, 1998); Medicago sativa, M63853 (Gowri et al, 1991); Nicotiana tabacum, X74452 (Jaeck et al, 1996); Ocimum basilicum, AF154918 ; Oryza sativa, XM480185 (unpublished data); Picea abies, AJ868575, unpublished; Populus tremuloides, X62096 ; Prunus amygdalus, X83217 (Garcia-Mas et al, 1995); Rosa chinensis, AJ439740 (Scalliet et al, 2002); Saccharum officinarum, AJ231133 (Selman-Housein et al, 1999); Sorghum bicolor, AY217766 (Bout and Vermerris, 2003); Triticum aestivum, AY226581 (Jang et al, 2003); Vanilla planifolia COMT, AY555144 (Pak et al, 2004); V. planifolia OMT-2, DQ400399; V. planifolia OMT-3, DQ400400; Zea mays, M73235 (Collazo et al, 1992). oxybenzene derivatives such as n-propyl gallate, ethyl gallate, and methyl gallate.…”

“…Site-directed mutagenesis and molecular modeling revealed that the distinct substrate specificities between the two C. breweri OMTs could be explained by differences in a few substrate binding residues. Similarly, two OMTs from Chrysosplenium americanum are 83% identical, yet one methylates both COMT substrates as well as some flavonoids and the other is inactive with COMT substrates but is active with a different group of flavonoids (Gauthier et al, 1996(Gauthier et al, , 1998. The C. americanum flavonoid OMT (FOMT) differs from the COMT at seven of the COMT substrate binding sites , suggesting that these residues are important for the observed substrate discrimination between the two enzymes.…”

Role of Serine 286 in cosubstrate binding and catalysis of a flavonolO-methyltransferase

“…U16794) and Ser286Arg cDNA clones were previously isolated and cloned in the pTRCHis vector (Invitrogen, Carlsbad) to produce a fusion protein containing a His-tag (Gauthier et al 1996). Sitedirected mutagenesis of specific amino acids was carried out using the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.), in accordance with manufacturer's instructions, and the FOMTx (Ser286Arg) clone was used as a template.…”

“…1). One of these clones, FOMT3′, was shown to encode the 3′/5′-OMT for 3,7,4′-trimethylquercetn (TMQ) (Gauthier et al 1996). Another clone, FOMTx, was identical to FOMT3′, except for a single aminoacid substitution at position 286 in the amino-acid sequence (Ser286Arg, Fig.…”