Functional Characterization And Catalytic Mechanism Of Key Enzymes Involving In Bibenzyl Biosynthesis From Liverworts

Bryophytes are very important taxa placed between algae and ferns. Liverworts produce a variety of natural metabolites including terpenoids and aromatic compounds, which collectively harbor broad chemical and bioactivity, such as antifungal, antifeedant, antioxidant, cytotoxic activity and anti-inflammatory. Thus liverworts are treasure trove for bioactive natural products. However, it is difficult to collect a sufficient biomass of active compounds because the liverworts usually small and grow mixed together in special and extreme environment. Molecular biology and metabolic engineering provide an option to get sufficient of interesting lead compound. A proposed bisbibenzyls biosynthesis pathway has been found in liverworts, though the enzymes involved in the biosynthesis pathway were scarcely investigated. Thus, isolation and characterization of the key enzyme of plant secondary metabolism, illustrating the biosynthetic pathways of important secondary metabolites is of great concern.Through screening cDNA library and transcriptome sequencing data, we got a number of genes involved in bisbibenzyls biosynthesis pathway from liverworts Plagiochasma appendiculatum and Marchantia paleacea. Several of them were studied in detail, including phenylalanine ammonia-lyase (PAL), the first key enzyme of phenylpropanoid biosynthesis pathway, the recombinant protein PaPAL exhibited high PAL activity and a lower TAL activity. Two cytochrome P450 genes were obtained, and the genomic structure and gene expression pattern responding to phytohome treatment were analyzed. Two types of STCSs (stilbenecarboxylate synthase, STCS) were cloned and heterologously expressed in Escherichia coli and the recombinant proteins showed different substrate selectivity. The crystal structure of STCS1 protein was obtained. The main research contents and results are as follows.1. Cloning and functional characteriation of phenylalanine ammonia-lyase (PAL)-the first key enzyme of bibenzyls biosynthesis pathway from liverwortsA fragment which showed high homology with phenylalanine ammonia-lyase (PAL) was obtained from the cDNA library of the liverwort P. appendiculatum, and the full length of PAL (designated as PaPAL) was cloned from both the cDNA and genomic DNA of P. appendiculatum. Bio informatics analysis showed that PaPAL’s predicted amino acid sequence shares more than 70% identity with PAL sequences reported in public databases, and contain the Ala-Ser-Gly catalytic triad and most other putative active site residues. In addition, the genomic DNA of PaPAL was found with no intron. The PaPAL protein was heterologously expressed in E. coli and the purified protein exhibited high PAL activity, catalyzing the conversion of L-phenylalanine to trans-cinnamic acid. However, the enzyme exhibited lower activity in catalyzing the formation of p-coumaric acid from L-tyrosine. Additionally, the PaPAL expression was induced by abiotic stress inducers methyl jasmonate and abscisic acid, thereby augmenting bisbibenzyls formation. These results suggest that PaPAL plays a key role in the early steps of bisbibenzyl biosynthesis and that abiotic stress can stimulate the expression of PaPAL, resulting in the accumulation of bisbibenzyls in the plant.2. Cloning and functional research of chalcone synthetase genes from liverworts4 genes putatively encoding chalcone synthetase were annotated in the transcriptome sequencing database. The full length cDNA of these genes were cloned from M. paleacea. The cDNA sequences showd high identity with MpSTCS1 and MpSTCS2 and were named MPaSTCSl-4. Sequence alignment showed that the STCSs sequences have a high homology with other CHSs, all containing the Cys-His-Asn catalytic triad and other conserved amino acid residues.The four STCSs can be divided into two types according to the enzyme activity in vitro. The four STCSs catalyze C6/C1 cyclization to generate phloretin with dihydro-p-coumaroyl-CoA as a starting substrate. The MPaSTCS2 protein was capable of catalyzing C6/C1 cyclization to produce naringenin with p-coumaroyl-CoA as the starting substrate, while the other three STCSs can not. The STCSs showed obvious selectivity with the two substrates, while the existing research results can not explain the difference. MPaSTCS1 was selected for further crystallographic studies to explain this difference from the structural point and further elucidate the catalytic mechanism.3. Preliminary crystallography of the STCS1 proteinHere the crystal structure of STCS1 with a 2.4A resolution was obtained. The STCS1 enzyme forms a symmetric dimer related by a two-fold crystallographic axis and each asymmetric unit contains six STCS1 molecules. Each STCS1 monomer consists of two structural domains and exhibits αβαβα pseudo-symmetric motif. The STCS1 homodimer contain two functionally independent active sites and each consists almost entirely of residues from a single monomer with an exception that the Met139 from the adjoining monmer. Crystallographic comparison of the STCS1 active site to that of MsCHS and MpSTCS2 reveals only minor differences in topology, which may lead to the substrate selectivity. The proposed interaction between substrate and product with amino acid residues of STCS1 molecule was analyzed and the results were compared with the crystal structures of CHS complexed with substrate and product analogs, with expectation to clarify the selectivity mechanism between STCS1 and CHS proteins.4. Cloning and function study of P450 genes related to the secondary metabolic pathwaysSeveral P450s in cDNA library of P. appendiculatum were proposed involved in bibenzyls biosynthesis. The full-length of two P450 genes designated as PaF3’H1 and PaF3’H2 were cloned and analyzed. There are two introns on the encoding region of the two P450 genes respectively. Bioinformatics analysis showed that they belong to the P450 superfamily with a high homology with CYP75 subfamily. They both contain the conserved domains of P450s, including the heme-binding domain FxxGxRxCxG and several other conserved domains or sequences of P450s. In addition, the expression of these two P450 was induced by hormones.