PURIFICATION AND IMMUNOLOGICAL CHARACTERIZATION OF A RECOMBINANT TRIMETHYLFLAVONOL 3'-O-METHYLTRANSFERASE

A flavonol O-methyltransferase cDNA clone (pF3'OMT) from Chrysospleniu m americanum was expressed in Escherichia coli Top 10 and the recombin ant protein was purified to near homogeneity by affinity chromatograph y on chelation resin and gel filtration on Superose 12 columns. The pu rified protein was enzymatically active as a 42kDa monomer and exhibit ed strict specificity for position 3' of 3,7,4'-trimethylquercetin. It did not accept the mono- or dimethyl analogs, the parent aglycone que rcetin or the phenylpropanoids, caffeic and 5-hydroxyferulic acids as substrates; thus indicating its involvement in the later steps of poly methylated flavonol synthesis in this plant. The K-m values of the enz yme for 3,7,4'-trimethylquercetin as substrate and S-adenosyl-L-methio nine as co-substrate were 7.2 and 20 mu M, respectively. The enzyme ac tivity was strongly inhibited by both Ni2+ and p-chloromercuribenzoate and was restored by the addition of EDTA or beta-mercaptoethanol, res pectively. Antibodies raised against the F3'OMT recombinant protein re cognized a protein band migrating at the expected molecular mass of th e enzyme on SDS-polyacrylamide gels of protein extracts prepared from various sources. This implies a high degree of structural similarity a mong these enzymes that is also corroborated by their hydropathy profi les. (C) 1998 Elsevier Science Ltd. All rights reserved.