Catalytic specificity of pea O-methyltransferases suggests gene duplication for (+)-pisatin biosynthesis

S-adenosyl-L-methionine: 2-hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) methylates 2,7, 4'-trihydroxyisoflavanone to produce formononetin, an essential intermediate in the synthesis of isoflavonoids with methoxy or methylenedioxy groups at carbon 4' (isoflavone numbering). HI4'OMT is highly similar (83% amino acid identity) to (+)-6a-hydroxymaackiain 3-O-methyltransferase (HMM), which catalyzes the last step of (+)-pisatin biosynthesis in pea. Pea contains two linked copies of HMM with 96% amino acid identity. In this report, the catalytic activities of the licorice HI4'OMT protein and of extracts of Escherichia coli containing the pea HMM1 or HMM2 protein are compared on 2,7,4'-trihydroxyisoflavanone and enantiomers of 6a-hydroxymaackiain. All these enzymes produced radiolabelled 2,7-dihydroxy-4'-methoxyisoflavanone or (+)-pisatin from 2,7,4'-trihydroxyisoflavanone or (+)-6a-hydroxymaakiain when incubated with [methyl-C-14]-S-adenosyl-L-methionine. No product was detected when (-)-6a-hydroxymaackiain was used as the substrate. HIWOMT and HMMI showed efficiencies (relative V-max/K-m) for the methylation of 2,7,4'-trihydroxyisoflavanone 20 and 4 times higher than for the methylation of (+)-6a-hydroxymaackiain, respectively. In contrast, HMM2 had a higher V-max and lower K-m on (+)-6a-hydroxymaackiain, and had a 67-fold higher efficiency for the methylation of (+)-6a-hydroxymaackiain than that for 2,7,4'-trihydroxyisoflavanone. Among the 15 sites at which HMMI and HMM2 have different amino acid residues, 11 of the residues in HMM1 are the same as found in HI4'OMTs from three plant species. Modeling of the HMM proteins identified three or four putative active site residues responsible for their different substrate preferences. It is proposed that HMM I is the pea HIWOMT and that HMM2 evolved by the duplication of a gene encoding a general biosynthetic enzyme (HIWOMT). (c) 2006 Elsevier Ltd. All rights reserved.