Kornblatt Jack, Muzac Ingrid, Lim Yoongho, Ahn Joong Hoon, Ibrahim Ragai K
Biology Department's Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, QC H4B 1R6, Canada.
Biochem Cell Biol. 2004 Oct;82(5):531-7. doi: 10.1139/o04-054.
O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3' encodes the 3'/5'-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3' by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3' with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3', no photoaffinity labeling could be achieved using [(14)CH(3)]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3' and Ser286Arg. Modeling FOMT3' on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 A from any of the residues of the active site or the AdoMet binding site of FOMT3'. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 A of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes.
O-甲基转移酶催化S-腺苷-L-甲硫氨酸的甲基基团转移至几类黄酮类化合物的特定羟基上。从美洲金腰子文库中分离得到的几个cDNA克隆中，FOMT3'编码部分甲基化黄酮醇的3'/5'-O-甲基化。另一个克隆FOMTx与FOMT3'仅相差一个氨基酸残基（Ser286Arg），其重组蛋白对所测试的任何黄酮类底物均无酶活性。用丙氨酸、亮氨酸、赖氨酸或苏氨酸取代FOMT3'中的Ser 286，几乎完全消除了O-甲基转移酶活性。与FOMT3'相反，使用[(14)CH(3)]AdoMet对突变重组蛋白无法实现光亲和标记，这表明Ser 286也是共底物结合所必需的。等温滴定量热法测量结果证实了这些结果。圆二色光谱排除了FOMT3'和Ser286Arg二级结构中任何显著的构象差异。根据查尔酮甲基转移酶的结构对FOMT3'进行建模表明，丝氨酸286与FOMT3'活性位点或AdoMet结合位点的任何残基距离均大于10 Å。同时，美洲金腰子的大多数OMT中282至290位残基是保守的。这些残基构成了亚基界面的很大一部分，其中至少五个残基与相对亚基的距离在4 Å以内。因此，似乎Ser286中的突变通过改变亚基之间的接触来发挥其影响，并且这些接触对于维持这组酶的AdoMet结合位点和活性位点的完整性是必要的。
