Abstract
Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.
Highlights
In plants, coumarins are synthesized via the general phenylpropanoid pathway[8]
F6’H1 consists of 361 amino acid residues and shows significant homology to other plant 2-oxoglutarate dependent dioxygenase (2OGD) such as anthocyanidin synthase from A. thaliana (34% identity), flavanone 3b-hydroxylase from
Diffraction analysis of the colorless plate shaped F6’H1 crystals indicated that they belonged to space group C2, with unit-cell parameters a = 193.22 Å b = 54.55 Å, c = 78.82 Å and γ = 111.5°
Summary
Specificity of Feruloyl-CoA received: 13 February 2015 accepted: 09 April 2015 Published: 20 May 2015. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. To better understand the mechanism of enzyme catalysis and substrate specificity, we generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis. The product 6’-hydroxyferuloyl-CoA is converted into a simple coumarin scopoletin via spontaneous trans/cis isomerization and lactonization (Fig. 1) This enzyme belongs to 2-oxoglutarate dependent dioxygenase (2OGD) family[10]. By comparison of two protein structures, we targeted two amino acid residues and verified their roles in enzyme activity and substrate selectivity by site-directed mutagenesis
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