Identification of 4-coumarate : coenzyme A ligase (4CL) substrate recognition domains

Ehlting, J Shin, JJK Douglas, CJ

J. Ehlting et al., Identification of 4-coumarate : coenzyme A ligase (4CL) substrate recognition domains, PLANT J, 27(5), 2001, pp. 455-465

35

INGLESE

Article

Plant Sciences","Animal & Plant Sciences

PLANT JOURNAL

0960-7412 → ACNP

27

5

2001

455 - 465

ISI

0960-7412(200109)27:5<455:IO4:CA>2.0.ZU;2-H

4-coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoi d pathway, provides precursors for the biosynthesis of a large variety of p lant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4-cournarate and other hydroxycinnamates in a two step reaction involving t he formation of an adenylate intermediate. 4 CL shares conserved peptide mo tifs with diverse adenylate-forming enzymes such as firefly luciferases, no n-ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid resid ues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address t his question, we took advantage of the difference in substrate usage betwee n the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzy mes 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 a bility upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish feru late utilization in the context of At4CL1. Sbd I corresponds to a region pr eviously identified as the substrate binding domain of the adenylation subu nit of bacterial peptide synthetases, while sbd II centers on a conserved d omain 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 s bd II, suggesting that these play roles in substrate recognition.