Identification, Molecular Cloning, and Functional Characterization of a Coniferyl Alcohol Acyltransferase Involved in the Biosynthesis of Dibenzocyclooctadiene Lignans in Schisandra chinensis

Abstract

Schisandra chinensis owes its therapeutic efficacy to the dibenzocyclooctadiene lignans, which are limited to the Schisandraceae family and whose biosynthetic pathway has not been elucidated. Coniferyl alcohol is the synthetic precursor of various types of lignans and can be acetylated to form coniferyl acetate by coniferyl alcohol acyltransferase (CFAT), which belongs to the BAHD acyltransferase family. This catalytic reaction is important because it is the first committed step of the hypothetical biosynthetic pathway in which coniferyl alcohol gives rise to dibenzocyclooctadiene lignans. However, the gene encoding CFAT in S. chinensis has not been identified. In this study, firstly we identified 37 ScBAHD genes from the transcriptome datasets of S. chinensis. According to bioinformatics, phylogenetic, and expression profile analyses, 1 BAHD gene, named ScBAHD1, was cloned from S. chinensis. The heterologous expression in Escherichia coli and in vitro activity assays revealed that the recombinant enzyme of ScBAHD1 exhibits acetyltransferase activity with coniferyl alcohol and some other alcohol substrates by using acetyl-CoA as the acetyl donor, which indicates ScBAHD1 functions as ScCFAT. Subcellular localization analysis showed that ScCFAT is mainly located in the cytoplasm. In addition, we generated a three-dimensional (3D) structure of ScCFAT by homology modeling and explored the conformational interaction between protein and ligands by molecular docking simulations. Overall, this study identified the first enzyme with catalytic activity from the Schisandraceae family and laid foundations for future investigations to complete the biosynthetic pathway of dibenzocyclooctadiene lignans.

Keywords: BAHD acyltransferase family; Schisandra chinensis; coniferyl alcohol; coniferyl alcohol acyltransferase; dibenzocyclooctadiene lignans.

Figure 1

Biosynthetic pathway of dibenzocyclooctadiene lignans. PAL: phenylalanine ammonia-lyase; C4H: cinnamate-4-hydroxylase; C3H: coumarate-3-hydroxylase; COMT: caffeic acid O-methyltransferase; 4CL: 4-hydroxycinnamate CoA ligase; CCR: cinnamoyl-CoA reductase; CAD: cinnamyl alcohol dehydrogenase; CFAT: coniferyl alcohol acyltransferase; and IGS1: isoeugenol synthase 1.

Figure 2

The TIC of the isoeugenol standard and the fruits of S. chinensis.

Figure 3

Maximum likelihood phylogenetic tree and conversed motif of 75 biochemically characterized BAHD acyltransferases and 37 putative ScBAHD acyltransferases. The five clades are shown in different colors.

Figure 4

Heat map exhibits expression patterns of 37 ScBAHD genes during the ripening stages of S. chinensis fruits, including the green stage (Green_1 and Geen_2), light red stage (Light red_1 and Light red_2), and dark red stage (Dark red_1 and Dark red_2). Red indicates high relative gene expression, whereas blue indicates low relative gene expression.

Figure 5

(A) SDS-PAGE analysis of recombinant ScCFAT. ScCFAT was separated using 12% SDS-PAGE gel electrophoresis and protein bands were visualized after staining with Coomassie brilliant blue. Lane 1: protein marker; Lane 2: purified ScCFAT. (B) In vitro analyses of ScCFAT and PhCFAT activities. (a) UPLC analysis of compounds found in a reaction mixture containing protein of pET-28a, coniferyl alcohol, and acetyl-CoA after 15 min of incubation. (b) UPLC analysis of compounds found in a reaction mixture containing purified PhCFAT, coniferyl alcohol, and acetyl-CoA after 15 min of incubation. (c) UPLC analysis of compounds found in a reaction mixture containing purified ScCFAT, coniferyl alcohol, and acetyl-CoA after 15 min of incubation. (d) UPLC analysis of coniferyl alcohol standard. (e) UPLC analysis of compounds found in a reaction mixture containing protein of pET-28a, cinnamyl alcohol, and acetyl-CoA after 15 min of incubation. (f) UPLC analysis of cinnamyl acetate standard. (g) UPLC analysis of compounds found in a reaction mixture containing purified ScCFAT, cinnamyl alcohol, and acetyl-CoA after 15 min of incubation. (h) UPLC analysis of cinnamyl alcohol standard.

Figure 6

Subcellular localization of ScCFAT. The fusion proteins were observed under a confocal laser scanning microscope. a, e show the green fluorescence channel; b, f show the chloroplast autofluorescence channel; c, g show the bright field channel; and d, h were created from the images shown in the first three panels. Scale bar = 20 μm.

Figure 7

(A) Homology modeling of ScCFAT structure constructed with Modeller. His¹⁵⁸ is shown in stick representation. (B) Enlarged views of molecular docking of ScCFAT with acetyl-CoA. (C) Enlarged views of molecular docking of ScCFAT with coniferyl alcohol.