Identification and Functional Characterization of Three Phenylalanine Ammonia-Lyase Genes from Fallopia multiflora (Thunb.) Harald

• Published in: Russian journal of bioorganic chemistry 2023-06, Vol.49 (3), p.655-663
• Main Authors: Zhengyang Yang, Yin, Minzhen, Cheng, Mingen, Chu, Shanshan, Zha, Liangping, Zhao, Yujiao, Peng, Huasheng
• Format: Article
• Language: English
• Subjects: Amino acids; Ammonia; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Cinnamic acid; Cloning; E coli; Gene expression; Genes; Life Sciences; Organic Chemistry; Phenylalanine; Roots; Traditional Chinese medicine
• ISSN: 1068-1620; 1608-330X
• DOI: 10.1134/S1068162023030263

Fallopia multiflora (Thunb.) Harald. is a commonly used traditional Chinese medicine that is rich in chemical constituents. Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the phenylalanine pathway, catalyzing the deamination of L-phenylalanine to generate trans-cinnamic acid. Three PALs ( FmPAL1 , FmPAL2 , and FmPAL3 ) were cloned and validated from F. multiflora for the first time, demonstrated the presence of a multigene family of PALs in F. multiflora . The predicted complete open reading frames (ORFs) of FmPAL1 , FmPAL2 , and FmPAL3 are 2118bp, 2109bp and 2160bp, encoding 705, 702 and 719 amino acids, respectively. The phylogenetic results indicated that FmPALs have significant evolutionary relatedness with the known PALs from dicotyledons. To further confirm their function, the three FmPALs were cloned into the pET-28a vector and expressed in Escherichia coli Trans BL21 (DE3). The enzymatic activities of the FmPALs recombinant proteins were assayed and showed that FmPALs have a role in the d-catalyzed conversion of L-phenylalanine to trans -cinnamic acid. All three FmPALs showed tissue-specific expression. The expression of FmPAL1 and FmPAL2 genes were highest in stems and lowest in roots. Differently, the expression of FmPAL3 gene was highest in leaves and lowest in roots.