Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase

• Published in: Structure (London) Vol. 23; no. 11; pp. 2032 - 2042
• Main Authors: Li, Zhi, Nair, Satish K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 03.11.2015; Elsevier
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Binding Sites; Coenzyme A Ligases - chemistry; Coenzyme A Ligases - metabolism; Magnesium - metabolism; Molecular Docking Simulation; Molecular Sequence Data; Nicotiana - enzymology; Plant Proteins - chemistry; Plant Proteins - metabolism; Protein Binding; Substrate Specificity
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2015.08.012

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 Mg2+ 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-Mg2+-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. •The Nt4CL2 adenylation conformation shows an ordered P loop and bound nucleotide•Structures of transient Nt4CL2-adenylate intermediate show substrate-binding pocket•The Nt4CL2 Val341 deletion renders the enzyme with sinapinate-utilizing capability The enzyme 4-coumarate:CoA ligase plays a fundamental role in the biosynthesis of plant secondary metabolite and is a central nexus to several branch pathways. Li and Nair present results of structural, biochemical, and enzyme engineering studies that will allow the use of this enzyme as a tool for biotechnology.