Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

Abstract

Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure-function relationships of this important family of proteins.

Keywords: 4-coumarate: CoA ligase; Peucedanum praeruptorum; biochemical characterization; biosynthesis mechanism; site-directed mutagenesis.

FIGURE 1

The proposed biosynthetic pathways of coumarins in P. praeruptorum. The primary for the biosynthesis of coumarins in P. praeruptorum are highlighted in light blue. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; C3H, 4-coumaric acid 3-hydroxylase; COMT, caffeic acid O-methyltransferase; 4CL, 4-coumarate: CoA ligase; C3′H, p-coumaroylshikimate/quinate 3-hydroxylase; F6′H, feruloyl CoA 6′-hydroxylase; CHS, chalcone synthase; C2′H, p-coumaroyl CoA 2′-hydroxylase.

FIGURE 2

Phylogenetic relationships between Pp4CLs and 4CLs from different plants. The neighbor-joining tree was constructed through MEGA 5.05 with 1000 replicates bootstrap support. The bootstrap values are marked at the branch points. The accession numbers of the proteins used for the preparation of this tree are listed in Supplementary Table S2.

FIGURE 3

Alignment of the amino acid sequences of Pp4CL1 and other plant 4CLs. The amino acid sequences shown are P. praeruptorum 4CL1 (Pp4CL1; KX254614), Arabidopsis thaliana 4CL1 (At4CL1; NP_001077697), Glycine max 4CL1 (Gm4CL1; NP_001236418.1), Populus tomentosa 4CL1 (Pt4CL1; AAL02144.1), Scutellaria baicalensis 4CL1 (Sb4CL1; BAD90936.1). The conserved peptide motifs Box I and II are highlighted. Black letters on pink background are designated as completely conserved residues. Black letters on blue background are designated as highly conserved residues. Residues proposed to function in 4CL substrate specificity are marked with triangles.

FIGURE 4

Expression profiles of Pp4CLs. (A) Tissue-specific expression of 17 putative 4CL genes. (B) Transcript abundance of three 4CL genes in roots at 0, 3, 6, 9, 12, and 24 h after MeJA treatment. (C) Expression level of three genes in P. praeruptorum with various treatments. “R” represents roots without any treatment. Values are means ± SE (n = 3).

FIGURE 5

Substrate specificity of recombinant Pp4CL1. HPLC analysis and Q-TOF-MS identification of reaction products generated by recombinant Pp4CL1 protein. (A) p-Coumaric acid as substrate. (B) Ferulic acid as substrate. (C) Caffeic acid as substrate. (D) Isoferulic acid as substrate. (E) o-Coumaric acid as substrate. (F) Cinnamic acid as substrate.

FIGURE 6

Subcellular localizations of P. praeruptorum 4CL1 and 4CL10. “Green” panels show GFP fluorescence, and chloroplast autofluorescence is shown in “Red” panels. “Merged” panels represent combined fluorescence from GFP and chloroplasts. Arabidopsis protoplasts containing empty vector were used as a control, namely 35S::GFP. 35S::Pp4CL1-GFP represents protoplasts containing pCAMBIA1302-4CL1 plasmid. Protoplasts containing pCAMBIA1302-4CL10 plasmid are abbreviated as 35S:: Pp4CL10-GFP. Bars = 20 μm.

FIGURE 7

Homology modeling and docking of Pp4CL1 with APP. (A) Three-dimensional model of Pp4CL1-APP complex. (B) The hydroxycinnamate binding pocket of Pp4CL1. (C) Stereoview of the Pp4CL1-APP interaction. (D) Two-dimensional model of Pp4CL1-APP complex. APP, adenosine 50-(3-(4-hydroxyphenyl) propyl) phosphate.

FIGURE 8

HPLC profile analysis of reaction products generated by mutations of Pp4CL1 (A) HPLC profile of mutations using p-coumaric acid as substrate. (B) HPLC profile of mutations using ferulic acid as substrate. (C) HPLC profile of mutations using caffeic acid as substrate. (D) HPLC profile of mutations using isoferulic acid as substrate. Numbers 0-12 represent: wild-type Pp4CL1 (0), Y239A (1), Y239F (2), Y239W (3), A243S (4), M306K (5), M306A (6), G308A (7), A309G (8), G334A (9), K441A (10), Q446A (11) and K526A (12).