Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase
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...
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Published in | Structure (London) Vol. 23; no. 11; pp. 2032 - 2042 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Ltd
03.11.2015
Elsevier |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 USDOE Office of Science (SC) |
ISSN: | 0969-2126 1878-4186 |
DOI: | 10.1016/j.str.2015.08.012 |