Poster: Secondary Metabolism

Abs # 271: The Substrate-Specificity Determining Amino Acid Code of 4-Coumarate:CoA Ligase

Presenter:	Kombrink, Erich , kombrink@mpiz-koeln.mpg.de
Authors	Kombrink, Erich  (A)   Schneider, Katja  (A)   Hövel, Klaus  (B)   Witzel, Kilian  (A)   Hamberger, Björn  (A)   Schomburg, Dietmar  (B)   Stuible, Hans-Peter  (A)
Affiliations:	(A): Max Planck Institute for Plant Breeding Research, Department of Plant Microbe Interactions, Carl-von-Linné-Weg 10, 50829 Köln, Germany (B): Institute of Biochemistry, University of Cologne, Zülpicher Straße 47, 50674 Köln, Germany

4-Coumarate:CoA ligase (4CL) is the branch point enzyme of general phenylpropanoid metabolism from which a large variety of plant secondary products is derived. Most 4CLs catalyze the conversion of the cinnamic acid derivatives coumarate, caffeate and ferulate to their corresponding CoA esters, but enzymes with different substrate utilization profiles have also been described. Arabidopsis 4CL2 (At4CL2) is unusual because its preferred substrate is caffeate, whereas ferulate and sinapate are not converted. To reveal the structural principles determining substrate specificity of 4CL, the crystal structure of the phenylalanine activation domain (PheA) of gramicidin S synthetase was used as a template for homology modeling. According to this model, twelve amino acid residues form the 4CL substrate binding pocket (SBP) and function as a signature motif determining substrate specificity. We utilized this specificity code to create At4CL2 gain-of-function mutants. Substitution of selected bulky amino acids by smaller residues resulted in enzymes that use ferulate instead of caffeate as preferred substrate. Deletion of selected amino acids generated sinapate activating At4CL2 variants. Substitution of charged or polar by hydrophobic amino acids strongly enhanced conversion of cinnamate. These results demonstrate that a size exclusion mechanism controls the accessibility of the At4CL2 SBP for mono- and dimethoxylated substrates, whereas activation of cinnamate is regulated by the hydrophobicity of the SBP. The new enzyme variants are suited tools to investigate and influence metabolic channeling mediated by 4CL. Knowledge of the 4CL substrate specificity code will facilitate the prediction of substrate preference of numerous uncharacterized Arabidopsis 4CL-like proteins.