Identification of the Substrate Specificity-conferring Amino Acid Residues of 4-Coumarate:Coenzyme A Ligase Allows the Rational Design of Mutant Enzymes with New Catalytic Properties

Abstract

4-Coumarate:coenzyme A ligases (4CLs) generally use, in addition to coumarate, caffeate and ferulate as their main substrates. However, the recently cloned Arabidopsis thaliana isoform At4CL2 is exceptional because it has no appreciable activity with ferulate. On the basis of information obtained from the crystal structure of the phenylalanine-activating domain of gramicidin S-synthetase, 10 amino acid residues were identified that may form the substrate binding pocket of 4CL. Among these amino acids, representing the putative "substrate specificity motif," only one residue, Met 293 , was not conserved in At4CL2, compared with At4CL1 and At4CL3, two isoforms using ferulate. Substitution of Met 293 or Lys 320 , another residue of the putative substrate specificity motif, which in the predicted three-dimensional structure is located in close proximity to Met 293 , by smaller amino acids converted At4CL2 to an enzyme capable of using ferulate. The activity with caffeate was not or only moderately affected. Conversely, substitution of Met 293 by bulky aromatic amino acids increased the apparent affinity (K m ) for caffeate up to 10-fold, whereas single substitutions of Val 294 did not affect substrate use. The results support our structural assumptions and suggest that the amino acid residues 293 and 320 of At4CL2 directly interact with the 3-methoxy group of the phenolic substrate and therefore allow a first insight into the structural principles determining substrate specificity of 4CL.

Molecular Cloning and Characterization of Genes Involved in Rosmarinic Acid Biosynthesis from <i>Prunella vulgaris</i>

“…Pv4CL had conserved motif (long bar-AMP binding domain, SSGTTGLPKGV; short bar-GEICIRG). 24,25) In addition, the phylogenetic tree showed that the Pv4CL sequence was homologous to other C4H sequences (Supplemental information Fig. S3).…”

The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes

“…1, all except sinapate are efficiently activated by numerous 4CLs from various sources, whereas sinapate has so far been converted with appreciable efficiency, apart from crude or partially purified protein extracts (2,5,(8)(9)(10), by just one pure heterologously expressed isoenzyme, Gm4CL1 from soybean (Glycine max L.) (11). A recent combination of structural, biochemical, and mutational analyses using At4CL2 from Arabidopsis thaliana as a test case revealed that the substrate-binding pocket of this and all other 4CLs with the same specificity-determining amino acid code is too small to accommodate sinapate (12,13). Directed mutations enlarging this pocket to a size similar to that realized in Gm4CL1 indeed led to the expected broadening of the substrate specificity, including the conversion of sinapate (13).…”

The Maize High-Lysine Mutantopaque7Is Defective in an Acyl-CoA Synthetase-Like Protein

“…The 4CL family in Arabidopsis has four members that are distantly related to 4CL-like proteins. However, both classes have two conserved box I and box II motifs, the amino acid residues in-between determining the substrate specificity of the enzyme (Stuible and Kombrink 2001;Schneider et al 2003). Both motifs can be identified in the O7 protein, with the 12-bp deletion of the o7-ref allele mapping between the two ( Figure 2B).…”

“…The o7-6 allele, on the other hand, abolished a lysine residue in the C-terminal domain as a consequence of the Ds insertion at amino acid 496. This lysine residue is important in adenylate formation (Stachelhaus et al 1999;Stuible and Kombrink 2001;Schneider et al 2003), thus probably causing an altered functionality of the O7-6 protein. At3g48990, which is the o7 homolog in Arabidopsis (Figure 5), grouped with yeast fatty acid CoA ligase and has been proposed to have the same function (Cukovic et al 2001).…”