Molecular Cloning, Biochemical Characterization, and Structural Insights into a Flavonoid-Associated Class II 4-Coumarate:CoA Ligase from Sageretia thea

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The enzymatic regulation of phenylpropanoid metabolism is a critical determinant of flavonoid biosynthesis in medicinal plants.Sageretia thea is valued for its pharmacological properties associated with flavonoid production; however, the molecular mechanisms governing pathway entry remain poorly understood.In this study, we identified and functionally characterized a novel 4-coumarate:CoA ligase (St4CL1) from S. thea.Phylogenetic analysis classified St4CL1 as a Class II isoform, a group typically associated with flavonoid biosynthesis, and multiple sequence alignment revealed the presence of highly conserved functional motifs, including the AMP-binding and catalytic domains.Recombinant St4CL1, heterologously expressed in Escherichia coli, exhibited a strong preference for Mg 2+ and optimal catalytic activity at pH 7.0-8.0and 35°C.Substrate specificity analysis revealed that St4CL1 exhibited the highest relative activity toward p-coumaric acid, supporting its role in directing carbon flux into the flavonoid biosynthetic pathway.Homology modeling and molecular docking revealed a conserved substrate-binding pocket within the interdomain cleft, and p-coumaric acid was the substrate to form a salt-bridge contact at the carboxylate-binding site, providing a structural rationale consistent with its preferred turnover.Collectively, these findings provide the first molecular evidence of the phenylpropanoid entry step in S. thea and identify St4CL1 as a promising enzymatic target for metabolic engineering to enhance flavonoid production.

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