Deletion of a single amino acid residue from different 4-coumarate:CoA ligases from soybean results in the generation of new substrate specificities.
Plant 4-coumarate:coenzyme A ligases, acyl-CoA ligases, peptide synthetases, and firefly luciferases are grouped in one family of AMP-binding proteins. These enzymes do not only use a common reaction mechanism for the activation of carboxylate substrates but are also very likely marked by a similar functional architecture. In soybean, four 4-coumarate:CoA ligases have been described that display different substrate utilization profiles. One of these (Gm4CL1) represented an isoform that was able to convert highly ring-substituted cinnamic acids. Using computer-based predictions of the conformation of Gm4CL1, a peptide motif was identified and experimentally verified to exert a critical influence on the selectivity toward differently ring-substituted cinnamate substrates. Furthermore, one unique amino acid residue present in the other isoenzymes of soybean was shown to be responsible for the incapability to accommodate highly substituted substrates. The deletion of this residue conferred the ability to activate sinapate and, in one case, also 3,4-dimethoxy cinnamate and was accompanied by a significantly better affinity for ferulate. The engineering of the substrate specificity of the critical enzymes that activate the common precursors of a variety of phenylpropanoid-derived secondary metabolites may offer a convenient tool for the generation of transgenic plants with desirably modified metabolite profiles.
Lindermayr
Christian
C
Department Biologie I/Botanik, Ludwig-Maximilians Universität, Menzinger Str. 67, D-80638 München, Germany.
Fliegmann
Judith
J
Ebel
Jürgen
J
D000595:Amino Acid Sequence; D000596:Amino Acids; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D007527:Isoenzymes; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase