Publications

• Partial purification, kinetic analysis, and amino acid sequence information of a flavonol 3-O-methyltransferase from Serratula tinctoria.

  Huang T.S., Anzellotti D., Dedaldechamp F., Ibrahim R.K.

  Serratula tinctoria (Asteraceae) accumulates mainly 3,3'-dimethylquercetin and small amounts of 3-methylquercetin as an intermediate. The fact that 3-methylquercetin rarely accumulates in plants in significant amounts, and given its important role as an antiviral and antiinflammatory agent that ac ... >> More

  Serratula tinctoria (Asteraceae) accumulates mainly 3,3'-dimethylquercetin and small amounts of 3-methylquercetin as an intermediate. The fact that 3-methylquercetin rarely accumulates in plants in significant amounts, and given its important role as an antiviral and antiinflammatory agent that accumulates in response to stress conditions, prompted us to purify and characterize the enzyme involved in its methylation. The flavonol 3-O-methyltransferase (3-OMT) was partially purified by ammonium sulfate precipitation and successive chromatography on Superose-12, Mono-Q, and adenosine-agarose affinity columns, resulting in a 194-fold increase of its specific activity. The enzyme protein exhibited an expressed specificity for the methylation of position 3 of the flavonol, quercetin, although it also utilized kaempferol, myricetin, and some monomethyl flavonols as substrates. It exhibited a pH optimum of 7.6, a pI of 6.0, and an apparent molecular mass of 31 kD. Its K(m) values for quercetin as the substrate and S-adenosyl-l-Met (AdoMet) as the cosubstrate were 12 and 45 microm, respectively. The 3-OMT had no requirement for Mg(2+), but was severely inhibited by p-chloromercuribenzoate, suggesting the requirement for SH groups for catalytic activity. Quercetin methylation was competitively inhibited by S-adenosyl-l-homo-Cys with respect to the cosubstrate AdoMet, and followed a sequential bi-bi reaction mechanism, where AdoMet was the first to bind and S-adenosyl-l-homo-Cys was released last. In-gel trypsin digestion of the purified protein yielded several peptides, two of which exhibited strong amino acid sequence homology, upon protein identification, to a number of previously identified Group II plant OMTs. The availability of peptide sequences will allow the design of specific nucleotide probes for future cloning of the gene encoding this novel enzyme for its use in metabolic engineering. << Less

  Plant Physiol 134:1366-1376(2004) [PubMed] [EuropePMC]

• Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. I. Partial purification and some properties of S-adenosyl-L-methionine:flavonol 3-, 6-, 7-, and 4'-O-methyltransferases.

  De Luca V., Ibrahim R.K.

  Four novel flavonol O-methyltransferases (OMTs) were partially purified from Chrysosplenium americanum by precipitation with ammonium sulfate, successive chromatography on Sephacryl S-200 and hydroxylapatite, and chromatofocusing on Polybuffer ion exchanger. They exhibited strict position specific ... >> More

  Four novel flavonol O-methyltransferases (OMTs) were partially purified from Chrysosplenium americanum by precipitation with ammonium sulfate, successive chromatography on Sephacryl S-200 and hydroxylapatite, and chromatofocusing on Polybuffer ion exchanger. They exhibited strict position specificity for positions 3 of quercetin, 7 of 3-methylquercetin, 4' of 3,7-dimethylquercetin, and 6 of 3,7,3'-trimethylquercetagetin. None of these enzymes reacted with phenylpropanoids, flavones, dihydroflavonols, or any of their glucosides. Except for the 7-OMT whose activity was lost during chromatofocusing, the other enzymes had apparent pI values of 4.8, 5.4, and 5.7 for the 3-, 4'-, and 6-OMTs, respectively. They had similar molecular weights (Mr 57,000) and their pH optima varied between 7.8 and 9.0, with a shift in optimal activity from lower to higher pH with increasing level of substrate methylation. Unlike the 3 and 4' enzymes, the 6-OMT showed an absolute requirement for Mg2+ whose activation was saturable and was inhibited by EDTA. The in vitro stepwise O-methylation of quercetin----3-methylquercetin----3,7-dimethylquercetin----3 ,7, 4'-trimethylquercetin tends to suggest a coordinated sequence of methyl transfers in this tissue. << Less

  Arch Biochem Biophys 238:596-605(1985) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. II. Substrate interaction and product inhibition studies of flavonol 3-, 6-, and 4'-O-methyltransferases.

  De Luca V., Ibrahim R.K.

  The steady-state kinetic behavior of three position-specific O-methyltransferases (3-, 4'-, and 6-OMTs) was compared with reference to substrate inhibition patterns in Chrysosplenium americanum. The 6-OMT was severely inhibited by the flavonoid substrate at concentrations close to Km, whereas the ... >> More

  The steady-state kinetic behavior of three position-specific O-methyltransferases (3-, 4'-, and 6-OMTs) was compared with reference to substrate inhibition patterns in Chrysosplenium americanum. The 6-OMT was severely inhibited by the flavonoid substrate at concentrations close to Km, whereas the other two enzymes were less affected by their respective flavonoid substrates. Substrate interaction kinetics for the 6-OMT gave converging lines consistent with a sequential binding mechanism, whereas the data generated for the 3- and 4'-OMTs could be fitted to the equation for a ping-pong mechanism or to that of a sequential binding mechanism where Kia was much smaller Ka. More information on the mechanism of reaction was obtained from product inhibition studies. The three enzymes exhibited competitive inhibition patterns between S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH), whereas other patterns were either noncompetitive or uncompetitive. The steady-state kinetic properties of the 3-, 4'-, and 6-OMTs were consistent with a sequential ordered reaction mechanism, in which SAM and SAH were leading reaction partners and included an abortive EQB complex. Product inhibition constants were sufficiently low to suggest that these may be important in regulating the pathway of polymethylated flavonoid synthesis. It was suggested that due to their greater sensitivity to inhibition by SAH, the OMTs involved in earlier steps of the methylation sequence may regulate the rate of synthesis of final products in Chrysosplenium. << Less

  Arch Biochem Biophys 238:606-618(1985) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.