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Canna~Fangled Abstracts

Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

By July 22, 2013No Comments

pm2Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

Source

Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195, Japan. yoshinari_shoyama@nmp.co.jp

Abstract

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6′ of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.
Copyright © 2012 Elsevier Ltd. All rights reserved.
PMID:

 22766313
[PubMed – indexed for MEDLINE]

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Highlights

► THCA synthase catalyzes the oxidative cyclization of CBGA into THCA. ► The structure-function relationship of THCA synthase was investigated. ► X-ray crystal structure of THCA synthase was determined to 2.75 Å resolution. ► Specific residues of THCA synthase were identified by mutational analysis.

Abbreviations

  • THCA, ∆1-tetrahydrocannabinolic acid;
  • CBGA, cannabigerolic acid;
  • FAD, flavin adenine dinucleotide;
  • BBE, berberine bridge enzyme;
  • THC, ∆1-tetrahydrocannabinol;
  • GOOX, glucooligosaccharide oxidase;
  • AknOx, aclacinomycin oxidoreductase;
  • PDB, Protein Data Bank

Keywords

  • THCA synthase;
  • X-ray crystallography

Figures and tables from this article:

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Fig. 1.

Oxidocyclization catalyzed by THCA synthase and the formula of products in this reaction.

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Fig. 2.

Relaxed stereo diagram of the tertiary structure of THCA synthase and the FAD binding site. (a) The overall structure of THCA synthase is shown in a ribbon model and α-helices are colored cyan, β-strands are in magenta, and loops are in pink. The FAD cofactor (blue) is shown in stick representation. The broken line between two domains (I and II) and the dotted line between two subdomains (Ia and Ib) delineate the general borders of these domains. (b) Stereo view of the tertiary structure and the Fo − Fc electron density map (cyan mesh; contoured at 2 σ level) for the FAD cofactor (cyan) and the covalently bound His114 and Cys176 (pink) in THCA synthase. In (b) and in Fig. 3, nitrogen, oxygen, phosphorus, and sulfur atom are colored blue, red, orange, and yellow, respectively.

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Fig. 3.

Relaxed stereo view of the key residues located near FAD of THCA synthase. (a) Locations of the ionizable residues (magenta) within ~ 15 Å (dashed circle) of the N5 atom of the isoalloxazine ring of FAD (cyan). The solvent-accessible surface was created using a probe radius of 1.4 Å. (a) is drawn as (b) in different orientation without surface. (c) Comparison of tyrosine and glutamic acid around the isoalloxazine ring of FAD between THCA synthase and similar enzymes (GOOX, orange; AnkOx, green; BBE, yellow). (c) is drawn in the same orientation as (b). All structures were superposed based on the isoalloxazine ring of FAD.

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Fig. 4.

Proposed catalytic mechanism of THCA synthesis from CBGA, based on the mutational analysis and the crystal structure of THCA synthase. Residues colored blue are from THCA synthase. FAD is colored black, and the substrate (CBGA) and product (THCA) are colored red.

Table 1.Enzymatic activities of the wild‐type and mutant THCA synthases
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Table 2.X-ray crystal, data collection, and refinement statistics
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Corresponding author contact information
Corresponding authors.
1
Present addresses: Yoshinari Shoyama, Research Center, Nihon Medi-Physics Co., Ltd., 3-1 Kitasode, Sodegaura, Chiba 299-0266, Japan; Yukihiro Shoyama, Faculty of Pharmaceutical Science, Nagasaki International University, Huis Ten Bosch-Cho 2825-7 Sasebo, Nagasaki 859-3298, Japan.

Copyright © 2012 Elsevier Ltd. Published by Elsevier Ltd. All rights reserved.

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