Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.
Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.
Hoffmann
L
L
Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Université Louis Pasteur, 12 rue du Général Zimmer, 67000 Strasbourg, France.
Maury
S
S
Bergdoll
M
M
Thion
L
L
Erard
M
M
Legrand
M
M
D000225:Adenine / Q000737:chemistry; D000486:Allergy and Immunology; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010947:Plants, Toxic; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D013912:Threonine / Q000737:chemistry; D014026:Nicotiana / Q000201:enzymology*
D011994:Recombinant Proteins; D005973:Glutamine; D013912:Threonine; D001120:Arginine; D008780:Methyltransferases; C059825:caffeoyl-CoA O-methyltransferase; D000225:Adenine