Shuai Gao’s research while affiliated with Shandong University and other places

Main conclusion The evolutionary conservation of type III polyketide synthases (PKS) in Selaginella has been elucidated, and the critical amino acid residues of the anther-specific chalcone synthase-like enzyme (SmASCL) have been identified. Abstract Selaginella species are the oldest known vascular plants and a valuable resource for the study of metabolic evolution in land plants. Polyketides, especially flavonoids and sporopollenin precursors, are essential prerequisites for plant land colonization. Although type III polyketide synthases (PKS) are widely studied in seed plants, the related enzymes in Selaginella remain poorly characterized. Here, eight type III PKSs were identified in the Selaginella moellendorffii genome and classified into three clusters. Two PKSs were selected for further research based on their phylogenetic relationships and protein sequence similarity. Functional studies revealed that they were chalcone synthase (SmCHS) and anther-specific CHS-like enzyme (SmASCL). These enzymes are involved in the biosynthesis of flavonoids and sporopollenin, respectively. Their sequence information and enzymatic activity are similar to the orthologs in other plants. Phylogenetic analysis revealed that the ASCL and CHS enzymes were separated into two clades from the Bryophyta. These results suggest that CHS and ASCL emerged in the first land plants and then remained conserved during plant evolution. To study the structural basis of the enzymatic function of SmASCL, a series of mutants were constructed. The number of condensation reactions catalyzed by the P210L/Y211D and I200V/G201T double mutants exceeds that of the wild-type enzyme. Our study provides insight into the characteristics and functions of type III PKSs in S. moellendorffii. It also offers clues for a deeper understanding of the relationship between active sites and the enzymatic function of ASCLs.

Phenylpropanoids play important roles in plant physiology and the enzyme 4-coumarate: coenzyme A ligase (4CL) catalyzes the formation of thioesters. Despite extensive characterization in various plants, the functions of 4CLs in the liverwort Marchantia paleacea remain unknown. Here, four 4CLs from M. paleacea were isolated and functionally analyzed. Heterologous expression in Escherichia coli indicated the presence of different enzymatic activities in the four enzymes. Mp4CL1 and Mp4CL2 were able to convert caffeic, p-coumaric, cinnamic, ferulic, dihydro-p-coumaric, and 5-hydroxyferulic acids to their corresponding CoA esters, while Mp4CL3 and Mp4CL4 catalyzed none. Mp4CL1 transcription was induced when M. paleacea thalli were treated with methyl jasmonate (MeJA). The overexpression of Mp4CL1 increased the levels of lignin in transgenic Arabidopsis. In addition, we reconstructed the flavanone biosynthetic pathway in E. coli. The pathway comprised Mp4CL1, co-expressed with chalcone synthase (CHS) from different plant species, and the efficiency of biosynthesis was optimal when both the 4CL and CHS were obtained from the same species M. paleacea.

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.

Vorinostat (suberoylanilide hydroxamic acid) was the first approved histone deacetylase (HDAC) inhibitor in a group of validated cancer therapeutic agents targeting epigenetics. Riluzole is a drug used to treat amyotrophic lateral sclerosis, the antitumor potency of which has been recently revealed. Herein, a novel hybrid of vorinostat and riluzole (compound 1) was rationally designed, synthesized, and evaluated. Compared with vorinostat, compound 1 exhibited superior total HDAC inhibitory activity and similar HDAC isoform selective profiles. The intracellular HDAC inhibition of compound 1 was confirmed by Western blot analysis. Moreover, compound 1 possessed more potent in vitro antiproliferative activity against all tested solid and hematological tumor cell lines than vorinostat. In vitro metabolic stability evaluation of compound 1 revealed better human plasma stability and comparable human liver microsomal stability than vorinostat. Additionally, compound 1 demonstrated more significant in vivo antitumor activity in a MDA-MB-231 xenograft model than vorinostat, which could be attributed to its superior in vitro antiproliferative activity and metabolic stability. Taken together, the results presented here support further research and development of compound 1 as a promising antitumor agent.

Background: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes. Results: In this study, we cloned and characterized the function of PabHLH1, a bHLH family protein encoded by the liverworts species Plagiochasma appendiculatum. PabHLH1 is phylogenetically related to the IIIf subfamily bHLHs involved in flavonoids biosynthesis. A transient expression experiment showed that PabHLH1 is deposited in the nucleus and cytoplasm, while the yeast one hybrid assay showed that it has transactivational activity. When PabHLH1 was overexpressed in P. appendiculatum thallus, a positive correlation was established between the content of bibenzyls and flavonoids and the transcriptional abundance of corresponding genes involved in the biosynthesis pathway of these compounds. The heterologous expression of PabHLH1 in Arabidopsis thaliana resulted in the activation of flavonoids and anthocyanins synthesis, involving the up-regulation of structural genes acting both early and late in the flavonoids synthesis pathway. The transcription level of PabHLH1 in P. appendiculatum thallus responded positively to stress induced by either exposure to UV radiation or treatment with salicylic acid. Conclusion: PabHLH1 was involved in the regulation of the biosynthesis of flavonoids as well as bibenzyls in liverworts and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT, EC: 2.3.1.133) is a key metabolic entry point for the synthesis of monolignols in vascular plants; however, little is known about HCT in liverworts. Here, the isolation and characterization of HCTs encoded by the two liverwort species, Plagiochasma appendiculatum and Marchantia paleacea, are described. The sequences of the two enzymes harbor features typical of BAHD family members, except for the presence of a stretch of >100 residues that are not represented in higher plant HCTs. When truncated versions of both genes, which were constructed to clarify the significance of these extra residues, were investigated, it became apparent that the full-length and the truncated gene products shared similar catalytic activity and recognized the same substrates in vitro. They also functioned equivalently in vivo either when transiently expressed in tobacco to cause a higher total production of CGA (5-CQA) and 4-CQA or stably expressed in liverworts to accumulate the lignin-like contents. A structural model of MpHCT suggests that its active site bind to its substrate similar to that of Arabidopsis thaliana HCT. While truncated forms of HCT were deposited in the nucleocytoplasm, the full-length versions occurred exclusively in the cytoplasm. The conclusion is that liverworts produce bona fide HCTs that represent a point of departure in studying the evolution of lignin synthesis in plants.

The over-expression of aminopeptidase N on diverse malignant cells is associated with the tumor angiogenesis and metastasis. In this report, one new series of leucine ureido derivatives containing the triazole moiety was designed, synthesized and evaluated as APN inhibitors. Among them, compound 13v showed the best APN inhibition with an IC50 value of 0.089 ± 0.007 μM, which was two orders of magnitude lower than that of bestatin (IC50 = 9.4 ± 0.5 μM). Compound 13v also showed dose-dependent anti-angiogenesis activities. Even at the lower concentration (10 μM), compound 13v presented similar anti-angiogenesis activity compared with bestatin at 100 μM in both the human umbilical vein endothelial cells (HUVECs) capillary tube formation assay and the rat thoracic aorta rings test. Moreover, compared with bestatin, 13v exhibited comparable, if not better in vivo anti-metastasis activity in a mouse H22 pulmonary metastasis model.

Liverworts, a section of the bryophyte plants which pioneered the colonization of terrestrial habitats, produce cyclic bisbibenzyls as a secondary metabolite. These compounds are generated via the phenylpropanoid pathway, similar with flavonoid biosynthesis for which bHLH transcription factors have been identified as one of the improtant regulators in higher plants. Here, a bHLH homolog (PabHLH) was isolated from the liverwort species Plagiochasma appendiculatum and its contribution to bisbibenzyl biosynthesis was explored. Variation in the abundance of PabHLH transcript mirrored that of tissue bisbibenzyl content in three different liverwort materials. A phylogenetic analysis based on the bHLH domain sequence suggested that the gene encodes a member of bHLH subgroup IIIf, which clusters proteins involved in flavonoid synthesis. The gene's transient expression in onion epidermal cells implied that its product localized to the nucleus and a transactivation assays in yeast showed that it was able to activate transcription. In both callus and thallus, the over-expression of PabHLH boosted bisbibenzyl accumulation, while also up-regulating PaPAL, Pa4CL1,PaSTCS1 and two genes encoding P450 cytochromes, and its RNAi-induced suppression down-regulated the same set of genes and reduced the accumulation of bisbibenzyls. The abundance of PaCHS and PaFNSI transcript was related to flavonoid accumulation in transgenic thallus. PabHLH represents a candidate for the metabolic engineering of bisbibenzyl content.

Chalcone synthases (CHSs) of the type III polyketide synthases (PKSs), catalyze the formation of a tetraketide intermediate from a CoA-tethered starter and malonyl-CoA but use different cyclization mechanisms to produce distinct chemical scaffolds. Herein, we characterized CHS and CHS-like enzymes (designated MpCHS and MpCHSL1, 2 and 3) from Marchantia paleacea and determined the crystal structure of MpCHSL1. MpCHS catalyzed a Claisen condensation to form chalcone, while MpCHSLs catalyzed the formation of lactonized α-pyrones in vitro. Based on the structural, mutational and in vitro biochemical analyses, we established that MpCHSL1 is structurally and functionally closer to prototype CHS than stilbene synthase, and characterized the structural basis for the functional diversity of the type III PKSs. A chalcone-forming mutant of MpCHSL1 was build directed by the structural information. These findings pave the way for future studies to elucidate the functional diversity of type III PKSs in liverwort.

The aerial organs of most terrestrial plants are covered by a hydrophobic protective cuticle. The main constituent of the cuticle is the lipid polyester cutin, which is composed of aliphatic and aromatic domains. The aliphatic component is a polyester between fatty acid/alcohol and hydroxycinnamoyl acid. The BAHD/HxxxD family enzymes are central to the synthesis of these polyesters. The nature of this class of enzymes in bryophytes has not been explored to date. Here, a gene encoding a fatty ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (HFT) has been isolated from the liverwort Marchantia emarginata and has been functionally characterized. Experiments based on recombinant protein showed that the enzyme uses ω-hydroxy fatty acids or primary alcohols as its acyl acceptor and various hydroxycinnamoyl-CoAs—preferentially feruloyl-CoA and caffeoyl-CoA—as acyl donors at least in vitro. The transient expression of a MeHFT-GFP fusion transgene in the Nicotiana benthamiana leaf demonstrated that MeHFT is directed to the cytoplasm, suggesting that the feruloylation of cutin monomers takes place there.