Structure–activity relationships of wheat flavone O‐methyltransferase – a homodimer of convenience

Abstract

Plants elaborate a bewildering variety of small molecules derived from the phenylpropanoid, alkaloid and isoprenoid biosynthetic pathways; these small molecules are frequently referred to as secondary metabolites. Further diversification of these metabolites is the result of a number of enzyme-catalyzed reactions, one of which is O-methylation [1].Flavone O-methyltransferase (FOMT) [NCBI Protein Database (NCBI) accession number DQ223971] from wheat is one of a class of proteins that carry out transmethylation from S-adenosyl-l-methionine (AdoMet) to the flavone tricetin ( Fig. 1) A significant feature that differentiates this FOMT from other O-methyltransferases is that it catalyzes the sequential methylation of tricetin, converting it to the 3¢-monomethyl derivative (selgin), the 3¢,5¢-dimethyl derivative (tricin), and finally the 3¢,4¢,5¢-trimethyl derivative (3¢,4¢,5¢-trimethyltricetin) [2]. Although plant enzymes of secondary metabolism are often viewed as rather nonspecific, three sequential methylations, by a single protein, at nonequivalent positions are not found in many instances. POMT-9, an O-methyltransferase from poplar, has been shown to catalyze the formation of three products from esculetin. In this case, there is formation of 6-Omonomethyl-esculetin, 7-O-monomethyl-esculetin, and 6,7-O-dimethyl-esculetin [7]; this differs from the FOMT described in this work, where the products Wheat flavone O-methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O-methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S-adenosyl-l-methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6-7 per minute. Even though the binding pockets for S-adenosyll-methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis.Abbreviations AdoMet, S-adenosyl-L-methionine; AUC, analytical ultracentrifugation; COMT, caffeic acid ⁄ 5-hydroxyferulic acid 3,5-O-methyltransferase; FOMT, flavone O-methyltransferase from wheat; ITC, isothermal titration calorimetry.

Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis

“…The fact that selgin does not accumulate neither in planta (unpublished data) nor in vitro enzyme assays [[4] and this work] indicates that selgin, the first methylated intermediate of tricetin methylation, does not leave the active site until completion of sequential methylation. Such a mechanism excludes channeling of the methylated intermediates from one active site to another, and is in agreement with a random enzyme reaction mechanism [28]. The stepwise methylation of tricetin starts at the 3'-hydroxyl group which is the preferred ( meta ) position for methylation because of its highest negative electron density [29], as previously shown with the classical examples: rat liver catechol OMT [19] and lignin monomers COMT [16], followed by methylation at the 5'-position, through re-orientation of the first methylated intermediate, selgin.…”

“…However, the significantly low level of methylation at the 4'-hydroxyl group of tricin (Additional file 4B) may be explained by (a) the weak binding affinity of the enzyme for tricin (data not shown), (b) the low negative electron density of this para hydroxyl group [29], (c) the steric hindrance caused by introduction of a bulky methyl group into the 4'-position of tricin, and/or (d) the competitive inhibition of the enzyme reaction by its final product, 3',4',5'-trimethyltricetin (ca < 5 μM) (unpublished data). In fact, the results of enzyme assays and HPLC analysis of the reaction products analysis support these assertions [4,28]. …”

Spatial organization of the vindoline biosynthetic pathway in Catharanthus roseus

“…5A-H and 6A-P, Table 1). Such homodimerization seems to be a common feature of plant O-methyltransferases and has been reported for other enzymes, such as flavone OMT (Kornblatt et al, 2008). Interestingly, the 16OMT homodimers are localized in the cytoplasm with exclusion from the nucleus (Fig.…”

Survey of the year 2008: applications of isothermal titration calorimetry

“…In 2008, we identified more than 500 articles after searching Web of Science, PubMed, SciDir and OVID databases using ‘isothermal AND titration AND calorimetry’ or ITC or ‘Isothermal Titration Calorimetry’ search terms. Following the format of previous annual surveys these have been classified into the following categories: References cited in the text and review articles 1–82. Protein–protein and protein–peptide interactions 83–222. Protein–small ligand or protein–drug interactions 223–321. Protein/peptide metal interactions 322–355. Protein/peptide nucleic acid interactions 356–387. Protein/peptide lipid interactions …”