Purification and immunological characterization of a recombinant trimethylflavonol 3'-O-methyltransferase.
A flavonol O-methyltransferase cDNA clone (pF3'OMT) from Chrysosplenium americanum was expressed in Escherichia coli Top 10 and the recombinant protein was purified to near homogeneity by affinity chromatography on chelation resin and gel filtration on Superose 12 columns. The purified protein was enzymatically active as a 42 kDa monomer and exhibited strict specificity for position 3' of 3,7,4'-trimethylquercetin. In did not accept the mono- or dimethyl analogs, the parent aglycone quercetin or the phenylpropanoids, caffeic and 5-hydroxyferulic acids as substrates; thus indicating its involvement in the later steps of polymethylated flavonol synthesis in this plant. The K(m) values of the enzyme for 3,7,4'-trimethylquercetin as substrate and S-adenosyl-L-methionine as co-substrate were 7.2 and 20 microM, respectively. The enzyme activity was strongly inhibited by both Ni2+ and rho-chloromercuribenzoate and was restored by the addition of EDTA or beta-mercaptoethanol, respectively. Antibodies raised against the F3'OMT recombinant protein recognized a protein band migrating at the expected molecular mass of the enzyme on SDS-polyacrylamide gels of protein extracts prepared from various sources. This implies a high degree of structural similarity among these enzymes that is also corroborated by their hydropathy profiles.
Seguin
J
J
Department of Biology, Concordia University, Montreal, Que., Canada.
Muzac
I
I
Ibrahim
R K
RK
D000818:Animals; D000917:Antibody Formation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D013379:Substrate Specificity
D010940:Plant Proteins; D011994:Recombinant Proteins; D008780:Methyltransferases; C114844:trimethylflavonol 3'-O-methyltransferase, Chrysosplenium americanum