Biological Characteristics of a Novel Bibenzyl Synthase (DoBS1) Gene from Dendrobium officinale Catalyzing Dihydroresveratrol Synthesis

Abstract

Bibenzyl compounds are one of the most important bioactive components of natural medicine. However, Dendrobium officinale as a traditional herbal medicine is rich in bibenzyl compounds and performs functions such as acting as an antioxidant, inhibiting cancer cell growth, and assisting in neuro-protection. The biosynthesis of bibenzyl products is regulated by bibenzyl synthase (BBS). In this study, we have cloned the cDNA gene of the bibenzyl synthase (DoBS1) from D. officinale using PCR with degenerate primers, and we have identified a novel type III polyketide synthase (PKS) gene by phylogenetic analyses. In a series of perfect experiments, DoBS1 was expressed in Escherichia coli, purified and some catalytic properties of the recombinant protein were investigated. The molecular weight of the recombinant protein was verified to be approximately 42.7 kDa. An enzyme activity analysis indicated that the recombinant DoBS1-HisTag protein was capable of using 4-coumaryol-CoA and 3 malonyl-CoA as substrates for dihydroresveratrol (DHR) in vitro. The Vmax and Km of the recombinant protein for DHR were 3.57 ± 0.23 nmol·min^-1·mg^-1 and 0.30 ± 0.08 mmol, respectively. The present study provides further insights into the catalytic mechanism of the active site in the biosynthetic pathway for the catalytic production of dihydroresveratrol by bibenzylase in D. officinale. The results can be used to optimize a novel biosynthetic pathway for the industrial synthesis of DHR.

Keywords: Dendrobium officinale; bibenzyl synthase; dihydroresveratrol; enzyme activity; in vitro biosynthesis.

Figure 1

The proposed biosynthetic pathway for bibenzyls. PAL: phenylalanine ammonia-lyase; C4H: cinnamic-4-hydroxylase; 4CL: 4-coumaryl-CoA ligase; DBR: double-bond reductases; BBS: bibenzyl synthase.

Figure 2

Amino acid sequence comparison of six type III PKSs of plant origin. A multiple sequence alignment was calculated with the DNAMAN package. Black shading indicates the homology of amino acids, while red and blue shading indicates amino acids with different similarity. The conserved catalytic residues in plant type III PKS (Cys164, His304 and Asn337, DoBS1 numbers) are represented by diamonds. Additionally, the highly conserved sequence G373FGPG (DoBS1 number) is indicated by red boxes.

Figure 3

The phylogenetic relationship of type III PKS. The numbers at the nodes represent the bootstrap. Source data are provided in the Supplementary Material titled “Construct Phylogenetic Tree Source Files”.

Figure 4

Figure of the pET-28a-DoBS1 recombinant plasmid construction and protein purification results. (a) Electrophoresis pattern of double-enzyme-digested recombinant plasmid. M: DNA marker (250–12,000 bp); Lane 1: results of double digestion of XbaI-XhoI empty vector; Lane 2: Results of double digestion of the pET-28a-DoBS1 recombinant plasmid. (b) DoBS1 was purified with the Ni²⁺-NTA column. M: 180 kDa Prestained Protein Marker; Lane 1: supernatant crude protein; Lane 2: the effluent obtained after incubating the supernatant crude protein with the equilibrated column packing for 1 h; Lane 3: equilibrate with 20 mM imidazole; Lane 4: washed with 50 mM imidazole; Lane 5: elution with 500 mM imidazole.

Figure 5

The results of the target protein verification. (a) Western blot analysis of the final puri-fied protein. M: 180 kDa predicted protein marker; Lane 1: Target protein. (b) The coverage of the identified peptides on the protein. (c) MS/MS analysis of peptide ion 1 (ATT-GEGLEWGVLFGFGPGLTVETVVLR). (d) MS/MS analysis of peptide ion 2 (NVEKCLEE-AFTPFGISDWNSIFWVPHPGGR).

Figure 6

Analysis of the enzymatic reaction products of the DoBS1 protein. (a) HPLC chromatograms of the catalytic reaction standard mix. (b) Blank group. (c) HPLC chromatogram of the reaction product.

Figure 7

Kinetic analysis of DoBS1 toward p-coumaroyl-CoA. The Michaelis-Menten equation, expressed as V = Vmax[S]/(Km + [S]). The calculated values for Vmax and Km of the DoBS1 pro-tein were obtained. An adjusted R-squared value of 0.99787 indicates an excellent fit of the curve to the observed value.