4-Coumareate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabilism, providing precursors for a variety of plant secondary metabolites such as flavonoids and lignin. The enzyme catalyzes formation of 4-coumaroyl-CoA by initial activation of 4-coumaric acid with ATP to form an acyl-AMP intermediate, which is followed by thioester bond formation with CoASH and the concomitant release of AMP. The activation of the carboxylate substrate with ATP to form the acyl-AMP intermediate is a common mechanism for other adenylate-forming enzymes, including fatty acyl-CoA synthetase, the adenylation domains of the modular non-ribosomal peptide synthetase, and firefly luciferase (the ANL superfamily enzymes). Here we report that 4CL from Arabiaopsis thaliana (At4CL2) shows remarkable substrate promiscuity and novel catalytic functions. First, it was demonstrated that, in the absence of CoASH, the enzyme catalyzed amide bond forming reactions by condensation of 4-coumaric acid and the amino group fo D- and L- amino acids. Second, the enzyme generated a variety of biologically active amides including homoserine lactones and capsaicin by coupling the corresponding acids and amines. Finally, unexpectedly, structure-based 4CL site-directed mutants, Y253F and Q345A, catalyzed formation of dipeptides from a series of amino acids in the presence of ATP. Thus, the CoA ligase was functionally converted to dipeptide synthetase by the single amino acid substitutions in the substrate binding pocket.
