Identification of 4-coumarate:coenzyme A ligase (4CL) substrate recognition domains.
4-coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoid pathway, provides precursors for the biosynthesis of a large variety of plant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4-coumarate and other hydroxycinnamates in a two step reaction involving the formation of an adenylate intermediate. 4 CL shares conserved peptide motifs with diverse adenylate-forming enzymes such as firefly luciferases, non-ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid residues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address this question, we took advantage of the difference in substrate usage between the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzymes convert 4-coumarate, only At4CL1 is also capable of converting ferulate. Employing a domain swapping approach, we identified two adjacent domains involved in substrate recognition. Both substrate binding domain I (sbd I) and sbd II of At4CL1 alone were sufficient to confer ferulate utilization ability upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish ferulate utilization in the context of At4CL1. Sbd I corresponds to a region previously identified as the substrate binding domain of the adenylation subunit of bacterial peptide synthetases, while sbd II centers on a conserved domain of so far unknown function in adenylate-forming enzymes (GEI/LxIxG). At4CL1 and At4CL2 differ in nine amino acids within sbd I and four within sbd II, suggesting that these play roles in substrate recognition.
Ehlting
J
J
Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
Shin
J J
JJ
Douglas
C J
CJ
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D017343:Genes, Plant; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase