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Cited by 25Plant biochemistry and biosynthesisPlant Gene Expression AnalysisPhysical Organic Chemistry and Reaction Kinetics

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Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY

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Kim, Colin Y., Mitchell, Andrew J., Kastner, David W., Albright, Claire E., Gutierrez, Michael A., Glinkerman, Christopher M., Kulik, Heather J., Weng, Jing-Ke·2023·Nature Communications
DOI 10.1038/s41467-023-36299-1PMC PMC9898226

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Abstract

Plants contain rapidly evolving specialized enzymes that support the biosynthesis of functionally diverse natural products. In coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered to accelerate coumarin formation as the only known BAHD enzyme to catalyze an intramolecular acyl transfer reaction. Here we investigate the structural and mechanistic basis for COSY’s coumarin synthase activity. Our structural analyses reveal an unconventional active-site configuration adapted to COSY’s specialized activity. Through mutagenesis studies and deuterium exchange experiments, we identify a unique proton exchange mechanism at the α-carbon of the o -hydroxylated trans -hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Quantum mechanical cluster modeling and molecular dynamics further support this key mechanism for lowering the activation energy of the rate-limiting trans -to- cis isomerization step in coumarin production. This study unveils an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on COSY’s evolutionary origin and its recruitment to coumarin biosynthesis in eudicots.

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Fig. 1 Structural and sequence characterization of COSY in comparison with HCT. a Scopoletin-bound AtCOSY at 2.5 Å resolution. The overall fold showcases a pseudo-symmetric N-terminal (residues 1–181; light pink) and C-terminal (residues 230–451; light blue) domains, connected by a linker loop (residues 182–229; yellow). The conserved BAHD catalytic residues (His161 and Trp371), along with product stabilizing residues (Phe40, Tyr42, and Cys164) are showcased within the active site pocket. Tetraethylene glycol (PG4) and acetate ion (ACT) are displayed. The mFo–DFc simulated annealing omit map with electron density map around SCO ligand is contoured at 1.0 σ. b Multiple sequence alignment of COSY orthologs from Solanum tuberosum, Glycine max, and A. thaliana with HCT orthologs from A. thaliana, Panicum virgatum and Coffea canephora. The full sequence alignment is shown in Supplementary Fig. 4. c Structural alignment of AtCOSY (green) and AtHCT (PDB: 5KJU; gray). The product of AtHCT, p-coumaroyl shikimate, is shown in yellow. Scopoletin is shown in purple. d Structural comparison between AtCOSY and AtHCT reveals an active site loop insertion (residues 390–406) in AtCOSY that occludes the corresponding acyl acceptor pocket in AtHCT.
Fig. 1 Structural and sequence characterization of COSY in comparison with HCT. a Scopoletin-bound AtCOSY at 2.5 Å resolution. The overall fold showcases a pseudo-symmetric N-terminal (residues 1–181; light pink) and C-terminal (residues 230–451; light blue) domains, connected by a linker loop (residues 182–229; yellow). The conserved BAHD catalytic residues (His161 and Trp371), along with product stabilizing residues (Phe40, Tyr42, and Cys164) are showcased within the active site pocket. Tetraethylene glycol (PG4) and acetate ion (ACT) are displayed. The mFo–DFc simulated annealing omit map with electron density map around SCO ligand is contoured at 1.0 σ. b Multiple sequence alignment of COSY orthologs from Solanum tuberosum, Glycine max, and A. thaliana with HCT orthologs from A. thaliana, Panicum virgatum and Coffea canephora. The full sequence alignment is shown in Supplementary Fig. 4. c Structural alignment of AtCOSY (green) and AtHCT (PDB: 5KJU; gray). The product of AtHCT, p-coumaroyl shikimate, is shown in yellow. Scopoletin is shown in purple. d Structural comparison between AtCOSY and AtHCT reveals an active site loop insertion (residues 390–406) in AtCOSY that occludes the corresponding acyl acceptor pocket in AtHCT.CC BY · PubMed Central
Fig. 2 In vitro coupled biochemical assay that examines the coumarin synthase activity of AtCOSY. a Reaction schematic of the conversion of 6-hydroxyferulic acid (6OHFA) to scopoletin using 4-coumarate-CoA ligase 1 from A. thaliana (At4CL1) and coumarin synthase (AtCOSY). b Combined LC-MS extracted ion chromatograms (XICs) of 165.05556 m/z and 209.04549 m/z; 1, and 191.03443 m/z; 2. As scopoletin production is spontaneous, assay carried out in absence of AtCOSY serves as the baseline control for the coumarin synthase activity measurement. 4CL1 + AtCOSY sample shows complete conversion of 6OHFA into scopoletin. c Total ion count of 6OHFA and scopoletin from LC-MS for the enzymatic assays in triplicates. Data are presented as mean values +/− standard error of the mean. Relevant quantitative data underlying this figure is provided as a Source data file. d Reconstructed pose of trans−6OHFCoA in the active site of the CoA-bound AtCOSY structure. Phe40, Tyr42, His161, Cys 164, Trp371, and backbone atoms of Gly166 are highlighted as they make close contact with the substrate. e Relative coumarin synthase activity of wild-type and mutant AtCOSY. Log2 fold-change between the LC-HRAM-MS peak areas of coumarin ([M-H]− = 191.03443 m/z) in 4CL1 + AtCOSY (WT or mutant) samples and that of 4CL1-only samples are shown. We performed the mutant assays for the following mutants (H161A, H161Q, W371H, W371A, W371V, W371M, Y42F, F40T, C164A, F40T/Y42S, Y42F/H161A, G166A, G166L, Y373A, Y373F, L374A, and “Loop-swap”). All assays were performed in triplicates and the error bars represent standard error of the mean (except for Y373A, which was performed in duplicate due to experimental error). Relevant quantitative data underlying this figure is provided as a Source data file. f Structural alignment of CoA-bound (blue) and scopoletin-bound (green) AtCOSY structures highlighting the dynamic loop containing Tyr373 and Leu374. Arrows indicate change in orientation of Tyr373 and Leu374 in CoA-bound structure compared to scopoletin-bound structure. Source data are provided as a Source Data file.
Fig. 2 In vitro coupled biochemical assay that examines the coumarin synthase activity of AtCOSY. a Reaction schematic of the conversion of 6-hydroxyferulic acid (6OHFA) to scopoletin using 4-coumarate-CoA ligase 1 from A. thaliana (At4CL1) and coumarin synthase (AtCOSY). b Combined LC-MS extracted ion chromatograms (XICs) of 165.05556 m/z and 209.04549 m/z; 1, and 191.03443 m/z; 2. As scopoletin production is spontaneous, assay carried out in absence of AtCOSY serves as the baseline control for the coumarin synthase activity measurement. 4CL1 + AtCOSY sample shows complete conversion of 6OHFA into scopoletin. c Total ion count of 6OHFA and scopoletin from LC-MS for the enzymatic assays in triplicates. Data are presented as mean values +/− standard error of the mean. Relevant quantitative data underlying this figure is provided as a Source data file. d Reconstructed pose of trans−6OHFCoA in the active site of the CoA-bound AtCOSY structure. Phe40, Tyr42, His161, Cys 164, Trp371, and backbone atoms of Gly166 are highlighted as they make close contact with the substrate. e Relative coumarin synthase activity of wild-type and mutant AtCOSY. Log2 fold-change between the LC-HRAM-MS peak areas of coumarin ([M-H]− = 191.03443 m/z) in 4CL1 + AtCOSY (WT or mutant) samples and that of 4CL1-only samples are shown. We performed the mutant assays for the following mutants (H161A, H161Q, W371H, W371A, W371V, W371M, Y42F, F40T, C164A, F40T/Y42S, Y42F/H161A, G166A, G166L, Y373A, Y373F, L374A, and “Loop-swap”). All assays were performed in triplicates and the error bars represent standard error of the mean (except for Y373A, which was performed in duplicate due to experimental error). Relevant quantitative data underlying this figure is provided as a Source data file. f Structural alignment of CoA-bound (blue) and scopoletin-bound (green) AtCOSY structures highlighting the dynamic loop containing Tyr373 and Leu374. Arrows indicate change in orientation of Tyr373 and Leu374 in CoA-bound structure compared to scopoletin-bound structure. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 In vitro coupled enzyme assay that examines the H/D exchange at C2 of the (D2)−6OHFA substrate. a Reaction schematic for the conversion of 3, (D2)−6-Hydroxyferulic-acid into 4, (D2)-Scopoletin by light and 5, (D1)-Scopoletin by COSY. b Combined LC-MS extracted ion chromatograms (XICs) of 167.06804 m/z; 3, 193.04747 m/z; 4 and 192.04114 m/z; 5. The colored circles represent the relevant substrate or product peaks in each XIC trace corresponding to the compound number in a. c H/D exchange rate of assays conducted with no AtCOSY, wild-type AtCOSY, and various AtCOSY mutants. H/D exchange rate is defined as the percentage of 5 out of the total ion intensity for m/z values corresponding to 4 and 5. All assays were performed with 4CL1 in duplicates with data presented as mean values. Relevant quantitative data underlying H/D exchange is provided as a Source Data file. d MS1 spectra of the relevant peaks from assays shown in b corresponding to compounds highlighted in a.
Fig. 3 In vitro coupled enzyme assay that examines the H/D exchange at C2 of the (D2)−6OHFA substrate. a Reaction schematic for the conversion of 3, (D2)−6-Hydroxyferulic-acid into 4, (D2)-Scopoletin by light and 5, (D1)-Scopoletin by COSY. b Combined LC-MS extracted ion chromatograms (XICs) of 167.06804 m/z; 3, 193.04747 m/z; 4 and 192.04114 m/z; 5. The colored circles represent the relevant substrate or product peaks in each XIC trace corresponding to the compound number in a. c H/D exchange rate of assays conducted with no AtCOSY, wild-type AtCOSY, and various AtCOSY mutants. H/D exchange rate is defined as the percentage of 5 out of the total ion intensity for m/z values corresponding to 4 and 5. All assays were performed with 4CL1 in duplicates with data presented as mean values. Relevant quantitative data underlying H/D exchange is provided as a Source Data file. d MS1 spectra of the relevant peaks from assays shown in b corresponding to compounds highlighted in a.CC BY · PubMed Central
Fig. 4 Computational insights into the proposed intermediates and reaction energetics of COSY-catalyzed coumarin synthesis. a A schematic of the proposed stepwise mechanism of COSY-catalyzed coumarin synthesis from 6OHFCoA. The reaction begins with deprotonation of the 6-OH of 6OHFCoA by His161, followed by delocalization of electrons through the conjugated double bond system involving C2 of 6OHFCoA. Then, Leu374 helps orient IM1 towards the catalytic water molecule through van der Waals force, leading to the addition of a proton at C2. The C2-C3 bond then undergoes isomerization from the s-trans to the s-cis configuration. Following isomerization, lactonization and release of CoA lead to the generation of scopoletin. The oxyanion of IM5 is depicted to be stabilized by a water molecule coordinated by Trp371, although the indole nitrogen of Trp371 may stabilize the oxyanion directly. Hydrogens that are relevant to stereoselective proton exchange are colored in red and blue. b The QM optimized large cluster model in the reactant state. Continuous chains of amino acids are shown as orange ribbons and included side chains are shown with gray carbons. The substrate is depicted with blue carbons. Hydrogen bonds are depicted as dotted purple lines. Relevant QM cluster model. xyz atomic coordinate file is provided as a Source data file. c The overall energetics for all intermediates of the proposed mechanism. Reactants are labeled as R and products are labeled as P. A putative transition state for the reaction mechanism in the absence of C2-protonation is illustrated in red.
Fig. 4 Computational insights into the proposed intermediates and reaction energetics of COSY-catalyzed coumarin synthesis. a A schematic of the proposed stepwise mechanism of COSY-catalyzed coumarin synthesis from 6OHFCoA. The reaction begins with deprotonation of the 6-OH of 6OHFCoA by His161, followed by delocalization of electrons through the conjugated double bond system involving C2 of 6OHFCoA. Then, Leu374 helps orient IM1 towards the catalytic water molecule through van der Waals force, leading to the addition of a proton at C2. The C2-C3 bond then undergoes isomerization from the s-trans to the s-cis configuration. Following isomerization, lactonization and release of CoA lead to the generation of scopoletin. The oxyanion of IM5 is depicted to be stabilized by a water molecule coordinated by Trp371, although the indole nitrogen of Trp371 may stabilize the oxyanion directly. Hydrogens that are relevant to stereoselective proton exchange are colored in red and blue. b The QM optimized large cluster model in the reactant state. Continuous chains of amino acids are shown as orange ribbons and included side chains are shown with gray carbons. The substrate is depicted with blue carbons. Hydrogen bonds are depicted as dotted purple lines. Relevant QM cluster model. xyz atomic coordinate file is provided as a Source data file. c The overall energetics for all intermediates of the proposed mechanism. Reactants are labeled as R and products are labeled as P. A putative transition state for the reaction mechanism in the absence of C2-protonation is illustrated in red.CC BY · PubMed Central
Fig. 5 Evolutionary origins of COSY in land plants. a Presence of COSY (pink circles) and F6′H1 (purple circles) orthologs among major lineages of land plants. This information is inferred from maximum-likelihood phylogenetic analyses of COSY and F6′H1 together with their homologs from representative plants with sequenced genomes (Supplementary Figs. 28 and 29). b Combined LC-MS XICs of 165.05556 m/z; 1, 209.04549 m/z; 1 and 191.03443 m/z; 2 showcasing the in vitro coumarin synthase activity of COSY orthologs from G. max (Soybean), A. thaliana (Arabidopsis), S. tuberosum (Potato), and S. bicolor (Sorghum). c Relative coumarin synthase activity of the four COSY orthologs quantified by the LC-HRAM-MS peak areas derived from the in vitro enzyme assay as shown in b. All assays were performed in triplicates. Data are presented as mean values with the error bars representing standard error of the mean.
Fig. 5 Evolutionary origins of COSY in land plants. a Presence of COSY (pink circles) and F6′H1 (purple circles) orthologs among major lineages of land plants. This information is inferred from maximum-likelihood phylogenetic analyses of COSY and F6′H1 together with their homologs from representative plants with sequenced genomes (Supplementary Figs. 28 and 29). b Combined LC-MS XICs of 165.05556 m/z; 1, 209.04549 m/z; 1 and 191.03443 m/z; 2 showcasing the in vitro coumarin synthase activity of COSY orthologs from G. max (Soybean), A. thaliana (Arabidopsis), S. tuberosum (Potato), and S. bicolor (Sorghum). c Relative coumarin synthase activity of the four COSY orthologs quantified by the LC-HRAM-MS peak areas derived from the in vitro enzyme assay as shown in b. All assays were performed in triplicates. Data are presented as mean values with the error bars representing standard error of the mean.CC BY · PubMed Central

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Metadata

Authors
Kim, C. Y., Mitchell, A. J., Kastner, D. W., Albright, C. E., Gutierrez, M. A., Glinkerman, C. M., Kulik, H. J., & Weng, J.-K.
Affiliations
  • Whitehead Institute for Biomedical Research (US)
  • Massachusetts Institute of Technology (US)
Corresponding author
Weng, Jing-Ke
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-02-03
Volume
14
Issue
1
Pages
597-597
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-36299-1
PMC
PMC9898226
OpenAlex
W4319078070
Cited by
25
References
1
Citations by year
2026: 7 · 2025: 5 · 2024: 8 · 2023: 4
FWCI
2.13
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (1745302)
  • Burroughs Wellcome Fund
  • W. M. Keck Foundation
  • Family Larsson‐Rosenquist Foundation
  • Arnold and Mabel Beckman Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant Gene Expression Analysis
  • Physical Sciences › Chemistry › Organic Chemistry › Physical Organic Chemistry and Reaction Kinetics
Keywords
COSY, coumarin biosynthesis, BAHD acyltransferase, trans-cis isomerization, lactonization, active site configuration, hydrogen-deuterium exchange, enzyme mechanism, eudicots
MeSH terms
Acyltransferases, Coumarins, Isomerism, Plants, Protons

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