Role of Serine 286 in cosubstrate binding and catalysis of a flavonol<i>O</i>-methyltransferase

Kornblatt, Jack A.; Muzac, Ingrid; Lim, Yoongho; Ahn, Joong Hoon; Ibrahim, Ragai K.

doi:10.1139/o04-054

Cited by 4 publications

(5 citation statements)

References 24 publications

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“…The situation is more complex when looking at residues that are not at the active site of the protein and are a few angstroms from the interface. In the case of flavonol 3′,5′‐ O ‐methyltransferase, mutation S286R which definitely does not involve an interfacial residue, results in almost complete loss of activity [24]. It would appear that Ser286 communicates with the active site of the opposing subunit across the interface.…”

Section: Discussionmentioning

confidence: 99%

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

2008

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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.

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Section: Discussionmentioning

confidence: 99%

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

2008

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“…In several instances, radiolabeled AdoMet and 8-azido AdoMet were shown to specifically label the MTase using UV radiation [6][7][8][9][10][11]. In addition, trypsin digestion of AdoMet-bound proteins followed by chromatography to establish the peptide/amino acid-AdoMet adduct and site-directed mutagenesis experiments of amino acid residues predicted to be associated with the AdoMet binding site demonstrated the requirement for one or more conserved residues (i.e., Tyr, Cys or Pro) or motifs in MTase specific activity [12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Photoaffinity Labeling and Mutational Analysis of 24‐C‐Sterol Methyltransferase Defines the AdoMet Binding Site

Jayasimha

Nes

2008

Lipids

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Photolabeling and site-directed mutagenesis were performed on recombinant Saccharomyces cerevisiae 24-C-sterol methyltransferase (SMT) to elucidate the location and role of active site residues involved in AdoMet binding and catalysis. Bioinformatic analysis of the SMT revealed a ten amino acid segment, conserved between L124 and P133, associated with the Rossmann-like fold belonging to AdoMet-dependent methyltransferases. Irradiation of the SMT in the presence of [methyl-3H3]AdoMet directly photolabeled the protein. The specificity of photolabeling was demonstrated by inactivation experiments with structural analogs of AdoMet, including sinefungin. Trypsin digestion of the [methyl-3H3]AdoMet photolabeled Erg6p afforded a single radioactive band in SDS-PAGE gel of 4 kDa. HPLC purification of this material generated a single radioactive fraction. The corresponding 3H-AdoMet-peptide adduct was subjected to Edman sequencing and the first fifteen residues gave a sequence Gly120-Asp-Leu-Val-Leu-Asp-Val-Gly-Cys-Gly-Val-Gly-Gly-Pro-Ala134 that contained the predicted AdoMet binding site. Amino acid residues in the tryptic digest fragment considered to bind covalently with the AdoMet at Asp125, Cys128, Pro133 and Tyr153 were replaced with leucine and analyzed kinetically and by photolabeling inactivation experiments. The results indicate that one or both of Cys128 and Pro133 are covalently bound to AdoMet.

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“…Substrate specificities of OMTs can not be accurately predicted on the basis of sequence similarity alone (Schroder et al 2002), and in some cases a single amino acid change can alter substrate specificity (Frick and Kutchan 1999;Gang et al 2002). Elucidation of the crystal structures of caffeoyl CoA 3-OMT and three different group I OMTs from alfalfa (Zubieta et al 2001Ferrer et al 2005) has now made it possible to explore the structural basis of OMT substrate specificity by homology-based modeling (Hoffmann et al 2001;Gang et al 2002;Kornblatt et al 2004;Yang et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula

et al. 2006

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Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism.

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

Cited by 4 publications

References 24 publications

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

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

Photoaffinity Labeling and Mutational Analysis of 24‐C‐Sterol Methyltransferase Defines the AdoMet Binding Site

Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula

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