Premium
Cations modulate the substrate specificity of bifunctional class I O ‐methyltransferase from Ammi majus
Author(s) -
Lukačin Richard,
Matern Ulrich,
Specker Silvia,
Vogt Thomas
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.10.032
Subject(s) - o methyltransferase , affinity chromatography , biochemistry , enzyme , methyltransferase , complementary dna , recombinant dna , escherichia coli , heterologous expression , open reading frame , biology , cofactor , phosphofructokinase 2 , substrate (aquarium) , chemistry , peptide sequence , methylation , dna , ecology , gene
Caffeoyl‐coenzyme A O ‐methyltransferase cDNA was cloned from dark‐grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli . The translated polypeptide of 27.1‐kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O ‐methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal‐affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation‐dependent O ‐methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg 2+ ‐ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg 2+ was replaced by Mn 2+ or Co 2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O ‐methyltransferases.