Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase / 生物工程学报

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.

Comparison of stilbene synthase from different plant sources for resveratrol biosynthesis / 生物工程学报

Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.

Crystal structures of plant uridine diphosphate-dependent glycosyltransferases / 生物工程学报

Glycosyltransferases (GTs) catalyze the transfer of a sugar residue of an activated sugar donor to an acceptor molecule. Many families 1 GTs utilize an uridine diphosphate (UDP) activated sugar as donor in the glycosylation reaction, and most of these belong to a group of GTs referred to as the UGTs. The relationship between the degree of amino acid sequence identity and substrate specificity of the plant UGTs is highly complicated, and the prediction of substrate specificity based on phylogenetic analyses need to be improved by more biochemical characterization. This review summarizes the three dimensional structures of plant UGTs published in the Protein Data Bank (PDB), including the detailed substrate interactions with the sugar and receptor binding pockets and mutational analyses of some critical amino acids. It will be helpful for biochemical characterization the substrate specificity of the individual UGT, and lay the foundation for the enzymatic and genetic manipulation of plant UGTs in the future.

Plant-specific type III polyketide synthases superfamily: crystal structures and functions / 生物工程学报

Plant type III polyketide synthase (PKS) generates backbones of a variety of plant secondary metabolites with diverse functions, and has long been models to elucidate the relationship between the three-dimensional structure and function. More than 80 type IIII PKS crystal structures with different functions have been reported in Protein Data Bank, including the crystal structures of the well-studied Chalcone Synthase of plant type III PKS, as well as the 6 other kinds of PKSs in the family, which are critical for understanding the structural basis for diverse starter molecule selectivity, polyketide chain length and the cyclization reaction. Structure-based analysis and site-directed mutagenesis are foundation for the investigation of enzyme engineering, genetic and metabolic engineering. This review summarized 7 plant-specific type III PKS in the aspects of their crystal structures and functions.