title: Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase.
abstract: Plant 4-coumarate:CoA ligase (4CL) serves as a central catalyst in the phenylpropanoid pathway that provides precursors for numerous metabolites and regulates carbon flow. Here, we present several high-resolution crystal structures of Nicotiana tabacum 4CL isoform 2 (Nt4CL2) in complex with Mg(2+) and ATP, with AMP and coenzyme A (CoA), and with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP. The Nt4CL2-Mg(2+)-ATP structure is captured in the adenylate-forming conformation, whereas the other structures are in the thioester-forming conformation. These structures represent a rare example of an ANL enzyme visualized in both conformations, and also reveal the binding determinants for both CoA and the hydroxycinnamate substrate. Kinetic studies of structure-based variants were used to identify residues crucial to catalysis, ATP binding, and hydroxycinnamate specificity. Lastly, we characterize a deletion mutant of Nt4CL2 that possesses the unusual sinapinate-utilizing activity. These studies establish a molecular framework for the engineering of this versatile biocatalyst.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: snair@illinois.edu.
authors.forename: Satish K
authors.initials: SK
authors.lastname: Nair
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology
chemical_list: D010940:Plant Proteins; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase; D008274:Magnesium