Structure and Molecular Mechanism of a Bacterial ADP-forming 4-Coumarate CoA Ligase
Debayan Chaudhury1* (firstname.lastname@example.org), Justin F. Acheson1, Ella R. Torkelson1, Kaya A. Meyers1, Yucen Pu3, Zimou Sun3, Marco Tonelli1, Craig A. Bingman1,2, Rebecca A. Smith1,2, Steven D. Karlen1,2, John Ralph1,2, Shawn D. Mansfield3, and Brian G. Fox1,2
1University of Wisconsin−Madison; 2Great Lakes Bioenergy Research Center; and 3University of British Columbia
Acyl-CoA ligases are enzymes that catalyze the adenosine triphosphate (ATP)-dependent conjugation of carboxylic acids to coenzyme-A (CoA) and enable the entry of these acids into metabolism as activated CoA thioesters. These CoA thioesters are important metabolic intermediates that feed carbon from the organic acids into a variety of anabolic and catabolic pathways allowing for the construction/destruction of diverse molecular scaffolds, thereby enabling cellular function. The aim of this project is the identification and biochemical characterization of acyl-CoA ligases from plant and microbial sources with the end goal of enabling precision metabolic engineering of the important bioenergy crop Populus trichocarpa (black cottonwood) with a focus on lignification.
4-Coumarate CoA ligases (4CLs) are a subset of acid-thiol ligases (E.C. 6.2.1.-) that catalyze the ATP-dependent thioesterification of 4-coumarate to CoA. In plants, these enzymes act upstream in the phenylpropanoid pathway and are therefore attractive targets for metabolic engineering as they provide control of metabolic flux over the downstream reactions that are rich in valuable and useful metabolites (Vogt 2010). 4CLs are also present in microbes where they function in aromatic degradation pathways which proceed through CoA thioester. The researchers report the structure and mechanism of a bacterial 4CL (ferA) from the ferulic degradation cluster of the lignin degrading bacterium Sphingomonas SYK-6 (Masai et al. 2002). This is the first crystal structure report of a conformation of bacterial 4-coumarate ligase. Catalysis in ferA proceeds via a reversible central metabolism-like ADP-forming mechanism through an acyl-phosphate intermediate instead of the AMP-forming mechanism widely reported for plant 4CLs, which proceeds through an acyl-adenylate intermediate. The co-crystal structure of ferA (2.46 Å, Rwork 22%, Rfree 25%) crystallized in the pre-ATP hydrolysis conformation bound to inorganic phosphate, feruloyl-CoA, and non-hydrolysable ATP analogue AMPPNP offers insight into the catalytic machinery and all the ligand binding sites on the ferA enzyme, and sheds light on the molecular interactions that enable enzyme specificity and catalysis. Researchers combine these structural insights with optical and NMR spectroscopic studies of the forward and reverse ligase reaction to elucidate the role of protein conformation and its intricate links to ligand binding and group transfer catalysis.
Vogt, T., 2010. “Phenylpropanoid Biosynthesis.” Molecular Plant 3(1), 2–20.
Masai, E., et al. 2002. “Cloning and Characterization of the Ferulic Acid Catabolic Genes of Sphingomonas paucimobilis SYK-6.” Applied and environmental microbiology 68(9), 4416–24
This work was supported by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under award no. DE-SC0020349 to B.G.F. C.A.B., R.A.S., S.D.K., S.D.M., and J.R. were also supported in part by the Great Lakes Bioenergy Research Center (GLBRC, DOE BER Office of Science DE-SC0018409). This study made use of the MS and NMR facilities at the GLBRC, and the National Magnetic Resonance Facility at Madison which is supported by NIH grant R24GM141526.