Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus.
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 Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ 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.
Lukacin
Richard
R
Institut für Pharmazeutische Biologie der Philipps-Universität Marburg, Deutschhausstr. 17A, D- 35037 Marburg/Lahn, Germany.
Matern
Ulrich
U
Specker
Silvia
S
Vogt
Thomas
T
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D031165:Ammi / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D008970:Molecular Weight; D009154:Mutation; D011794:Quercetin / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; C058645:caffeoyl-coenzyme A; D011794:Quercetin; D008780:Methyltransferases; D008274:Magnesium