Biochemical Characterization Of Key Enzymes In Flavonoids Biosynthesis Pathyway From Liverworts

Bryophytes are a class of widely distributed important plant groups, which propagated by spore, transited from aquatic to terrestrial. Because the growing environment is hostile, extremely abundant secondary metabolites produced in the body of bryophytes. These secondary metabolites which possess a variety of biological activities can be used as lead compounds for new drug development, so it is known as a source of biologically active substances. Secondary metabolites in liverworts mainly include terpenes, bibenzyls and flavonoids.Flavonoids are low molecular weight polyphenolic phytochemicals which present in plants widely. Flavonoids are the main medicinal ingredients in some medicinal plants, and exist in some fruits, vegetables and drinks. Flavonoids which exist in plants play a key role in regulating the growth and development of plants, resisting external abiotic stresses and insect infestation. Various flavonoids have been evaluated for several biological activities like anti-inflammatory, anti-bacterial, anti-allergic, anti-oxidant and anti-cancer, etal. The biosynthetic pathway of flavonoids in higher plants has been studied extensively and was proved derived from phenylpropane and malonic acid pathway. However, due to the lack of genomic information, the biosynthetic pathway of flavonoids in liverworts was studied less. In our laboratory, the cDNA library of the Plagiochasma appendiculatum and the transcriptome sequencing of Marchantia paleacea were constructed. In this paper, two genes, showing high homology with chalcone isomerase (CHI), were found from the databases of two liverworts, and named as PaCH11, PaCH12, MpaCH11 and MpaCHI2, respectively. They were ligated into prokaryotic expression vector, expressed in BL21(DE3) and purified proteins for enzyme characterization in vitro. The chiral of enzyme products with naringenin chalcone as substrate were analyzed in order to identify the stereoscopic selectivity of chalcone isomerase. Enzyme activities results showed that PaCHIl and MpaCHI1 could catalyze the intramolecular cyclization of naringenin chalcone and isoliquiritigenin into (2S)-naringenin and (2S)-liquiritigenin, respectively. This is the first time that type Ⅱ CHIs were found in non-legume plants. However, PaCHI2 and MpaCHI2 lacked chalcone-to-flavanone catalytic ability. The phylogenetic analysis showed that PaCHI2 and MpaCHI2 were classified as a cluster with the typeⅣ chalcone isomerase (CHI-like), while PaCHI1 and MpaCHI1 were classified as a class with the type Ⅰ and type Ⅱ chalcone isomerase. According to the evolutionary relationships, PaCHIl and MpaCHIl were close to the type Ⅱ chalcone isomerase. The chiral analysis of enzyme products showed that PaCHI1 and MpaCHI1 had stereoselectivity which generate 2S-flavonoids.The flavone synthase played a key role in the biosynthetic pathway. In this paper, a gene which had a high homology with flavone synthase I was screened from the cDNA library of the P. appendiculatum, named PaFNS Ⅰ. The full-length sequence was cloned using 5’-RACE method and ligated into prokaryotic expression vector. Purified proteins were used for enzyme characterization. The analysis of enzyme activity with naringenin as substrate showed that the enzyme catalyzed the conversion of naringenin to 2-hydroxynaringenin and apigenin. The result implied that PaFNS Ⅰ exhibited both flavone synthase and flavanone 2-hydroxylase activity. The analysis of enzyme products, provided with dihydrokaempferol as substrate, showed that the protein had a weak flavonol synthase (FLS) function. However PaFNS Ⅰ did not show any catalytic activity for eriodictyol and dihydroquercetin. According to the sequence alignment and protein molecular modeling, four key amino acids Ala120, Phe146, Tyr240 and Leu31 were ultimately determined to be mutated to Met120, He146, Pro240 and Phe311, respectively. The enzyme activity of mutants showed that when the Y240 was mutated to P240, the mutant could converted naringenin to apigenin without producing any 2-hydroxynaringenin. Our assumption is that the phenol ring of Tyr240 forms a hydrophobic π-π stacking interaction with the naringenin A-ring, once this effect was damaged, the generation of 2-hydroxynaringenin would hindered. The Y240 was also mutated into Ala, Val, Ile and Phe. In addition, Ala120, Phe146, Tyr240 and Leu311 mutations were combined with each other for site-directed mutation. Comprehensive analysis of the ultimately enzyme activity of all mutants showed that Y240 was critical for 2-hydroxynaringenin formation. This is the first time that a FNS I with flavanone 2-hydroxylase activity has been reported and the key amino acids which determined the ratio of products was found out.