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Molecular analysis of type III polyketide synthase (PKS) gene family from Zingiber officinale Rosc
Abstract
Enzymes of the type III polyketide synthase family is considered to have significant role in biosynthesis of structurally diverse polyketide scaffolds in Zingiber officinale. Genome wide analysis of polyketide synthase gene family in Z. officinale identified partial sequences of six members. Comparative sequence analysis showed that four of them ZoPKS2, 3, 4 and 6 were novel forms as revealed by the significant variations at the highly conserved regions. Phylogenetic analysis also showed separate clustering of the novel forms along with the non-chalcone forming PKSs. This sequence identified in the study forms the first and basic information of the PKS gene family from the Zingiberaceae.
... In the typical enzymatic reaction, PKS forms the chalcone by stepwise decarboxylative condensation of one coumaroyl CoA molecule with three malonyl CoA moieties, followed by a Claisen type cyclization of the tetraketide product (Ferrer et al., 1999). The amazing biochemical diversity of PKSs is shown by the identification and characterization of expanding members of the family, such as the 2-pyrone synthase, stilbene synthase, benzalacetone synthase, valerophenone synthase, acridon synthase, etc. (Austin and Noel, 2003, Radhakrishnan et al., 2009). But considering the huge metabolic complexity of plants, more members are very likely to occur in plants, especially in medicinal plants. ...
... Most of the genes coding for the biosynthetic machinery of plant secondary metabolism are encoded by small families of genes originated through duplication over evolutionary time (Durbin et al., 2000). Type III polyketide synthase or chalcone synthase occurs in most plants as multigene families (Radhakrishnan and Soniya, 2009). In legumes, it forms multigene family of 6 to 12 members (Tuteja et al., 2004; Matsumura et al., 2005). ...
Plant phenolic compounds form a valuable resource of secondary metabolites having a broad spectrum of biological activities. Type III polyketide synthases play a key role in the formation of basic structural skeleton of the phenolic compounds. As a group of medicinal plants, PKSs with novel features are expected in the genome of Zingiberaceae. The genomic exploration of PKS in Alpinia calcarata conducted in this study identified the presence of an unusual intron at the region forming the second exon of typical PKSs, forming a gateway information of distribution of novel PKSs in Zingiberaceae.
... [35] Type III polyketide synthase (PKS) or chalcone synthase is a multigene family available in most of the plants. [38] A highly conserved stretches of amino acid residues of Polyketide synthases form catalytic pockets. The amino acid residues forming these pockets are located mostly within the second exon. ...
Alpinia calcarata Roscoe (Family: Zingiberaceae), is a rhizomatous perennial herb, which is commonly used in the traditional
medicinal systems in Sri Lanka. Alpinia calcarata is cultivated in tropical countries, including Sri Lanka, India, and Malaysia.
Experimentally, rhizomes of Alpinia calcarata are shown to possess antibacterial, antifungal, anthelmintic, antinociceptive,
anti‑inflammatory, antioxidant, aphrodisiac, gastroprotective, and antidiabetic activities. Phytochemical screening revealed
the presence of polyphenols, tannins, flavonoids, steroid glycosides and alkaloids in the extract and essential oil of this plant.
Essential oil and extracts from this plant have been found to possess wide range of pharmacological and biological activities.
This article provides a comprehensive review of its ethnomedical uses, chemical constituents and the pharmacological
profile as a medicinal plant. Particular attention has been given to the pharmacological effects of the essential oil of Alpinia
calcarata in this review so that the potential use of this plant either in pharmaceutics or as an agricultural resource can
be evaluated.
... Families of PKS genes have been reported in many plants, such as Daucus carota L. (Hirner and Seitz, 2000), Gerbera hydrida (Helariutta et al., 1996), Glycine max (Shimizu et al., 1999), Humulus lupulus (Novak et al., 2006), Hypericum androsaemum (Liu et al., 2003), Ipomoea purpurea (Durbin et al., 2000), Lycopersicon esculentum (O'Neill et al., 1990), Medicago sativa L. (Junghans et al., 1993), Petunia hybrida (Koes et al., 1989), Phaseolus vulgaris (Ryder et al., 1987), Pinus sylvestris L. (Preisig-Muller et al., 1999), Pisum sativum (Harker et al., 1990), Psilotum nudum (Yamazaki et al., 2001), Rheum palmatum (Abe et al., 2005), Rubus idaeus (Kumar and Ellis, 2003), Ruta graveolens (Springob et al., 2000), Saccharum spp. (Contessotto et al., 2001), Sorghum bicolor (Lo et al., 2002), Vitis vinifera (Wiese et al., 1994;Goto-Yamamoto et al., 2002), and Zingiber officinale (Radhakrishnan and Soniya, 2009). Their expression is controlled differently and it has been suggested that PKSs have evolved by gene duplication and, subsequently, divergence by mutations, providing an adaptative differentiation to plants (Tropf et al., 1994;Durbin et al., 2000;Lukacin et al., 2001). ...
Cannabinoids, flavonoids, and stilbenoids have been identified in the annual dioecious plant Cannabis sativa L. Of these, the cannabinoids are the best known group of this plant's natural products. Polyketide synthases (PKSs) are responsible for the biosynthesis of diverse secondary metabolites, including flavonoids and stilbenoids. Biosynthetically, the cannabinoids are polyketide substituted with terpenoid moiety. Using an RT-PCR homology search, PKS cDNAs were isolated from cannabis plants. The deduced amino acid sequences showed 51%-73% identity to other CHS/STS type sequences of the PKS family. Further, phylogenetic analysis revealed that these PKS cDNAs grouped with other non-chalcone-producing PKSs. Homology modeling analysis of these cannabis PKSs predicts a 3D overall fold, similar to alfalfa CHS2, with small steric differences on the residues that shape the active site of the cannabis PKSs.
With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.
Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclizes. The crystal structures of CHS alone and complexed with substrate and product analogs reveal the active site architecture that defines the sequence and chemistry of multiple decarboxylation and condensation reactions and provides a molecular understanding of the cyclization reaction leading to chalcone synthesis. The structure of CHS complexed with resveratrol also suggests how stilbene synthase, a related enzyme, uses the same substrates and an alternate cyclization pathway to form resveratrol. By using the three-dimensional structure and the large database of CHS-like sequences, we can identify proteins likely to possess novel substrate and product specificity. The structure elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.
Oxidative modification of LDL is thought to play a key role in the pathogenesis of atherosclerosis. Consumption of nutrients rich in phenolic antioxidants has been shown to be associated with attenuation of development of atherosclerosis. This study was undertaken to investigate the ex vivo effect of standardized ginger extract on the development of atherosclerosis in apolipoprotein E-deficient (E(0)) mice, in relation to plasma cholesterol levels and the resistance of their LDL to oxidation and aggregation. E(0) mice (n = 60; 6-wk-old) were divided into three groups of 20 and fed for 10 wk via their drinking water with the following: group i) placebo (control group), 1.1% alcohol and water (11 mL of alcohol in 1 L of water); group ii) 25 microg of ginger extract/d in 1.1% alcohol and water and group iii) 250 microg of ginger extract/day in 1.1% alcohol and water. Aortic atherosclerotic lesion areas were reduced 44% (P<0.01) in mice that consumed 250 microg of ginger extract/day. Consumption of 250 microg of ginger extract/day resulted in reductions (P<0.01) in plasma triglycerides and cholesterol (by 27 and 29%, respectively), in VLDL (by 36 and 53%, respectively) and in LDL (by 58 and 33%, respectively). These results were associated with a 76% reduction in cellular cholesterol biosynthesis rate in peritoneal macrophages derived from the E(0) mice that consumed the high dose of ginger extract for 10 wk (P<0.01). Furthermore, peritoneal macrophages harvested from E(0) mice after consumption of 25 or 250 microg of ginger extract/day had a lower (P<0.01) capacity to oxidize LDL (by 45 and by 60%, respectively), and to take up and degrade oxidized LDL (by 43 and 47%, respectively). Consumption of 250 microg of ginger extract/day also reduced (P<0.01) the basal level of LDL-associated lipid peroxides by 62%. In parallel, a 33% inhibition (P<0.01) in LDL aggregation (induced by vortexing) was obtained in mice fed ginger extract. We conclude that dietary consumption of ginger extract by E(0) mice significantly attenuates the development of atherosclerotic lesions. This antiatherogenic effect is associated with a significant reduction in plasma and LDL cholesterol levels and a significant reduction in the LDL basal oxidative state, as well as their susceptibility to oxidation and aggregation.
Curcuminoids, major components of the spice turmeric, are used as a traditional Asian medicine and a food additive. Curcumin, a representative curcuminoid, has received a great deal of attention because of its anti-inflammatory, anticarcinogenic, and antitumor activities. Here we report a novel type III polyketide synthase named curcuminoid synthase from Oryza sativa, which synthesizes bisdemethoxycurcumin via a unique mechanism from two 4-coumaroyl-CoAs and one malonyl-CoA. The reaction begins with the thioesterification of the thiol moiety of Cys-174 by a starter molecule, 4-coumaroyl-CoA. Decarboxylative condensation of the first extender substrate, malonyl-CoA, onto the thioester of 4-coumarate results in the formation of a diketide-CoA intermediate. Subsequent hydrolysis of the intermediate yields a beta-keto acid, which in turn acts as the second extender substrate. The beta-keto acid is then joined to the Cys-174-bound 4-coumarate by decarboxylative condensation to form bisdemethoxycurcumin. This reaction violates the traditional head-to-tail model of polyketide assembly; the growing diketide intermediate is hydrolyzed to a beta-keto acid that subsequently serves as the second extender to form curcuminoids. Curcuminoid synthase appears to be capable of the synthesis of not only diarylheptanoids but also gingerol analogues, because it synthesized cinnamoyl(hexanoyl)methane, a putative intermediate of gingerol, from cinnamoyl-CoA and 3-oxo-octanoic acid.
A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.
The biosynthesis of [6]-gingerol has been investigated by the administration of labelled precursors to whole Zingiber officinale plants and to rhizome sections. The results show that phenylalanine is elaborated to ferulic acid which then condenses, in an unusual version of the ‘biological Claisen’ reaction, with malonate and hexanoate to yield [6]-dehydrogingerdione (11). Hexanoate is incorporated intact as shown by the location of label and the constancy of isotope ratios. Dihydroferulate is accepted, but loss of tritium shows it to be first dehydrogenated. Dione (11) is reduced in two steps to [6]-gingerol; in the preferred path, CO reduction precedes CC reduction, with (12) as intermediate, although dione (13) is also converted.
Thesensitivity ofthecommonlyusedprogressive multiple sequence alignment methodhasbeengreatly improved forthealignment ofdivergent protein sequences. Firstly, individual weights areassigned to eachsequence inapartial alignment inorder todown- weightnear-duplicate sequences andup-weight the mostdivergent ones. Secondly, aminoacid substitution matrices arevaried atdifferent alignment stages according tothedivergence ofthesequences tobe aligned. Thirdly, residue-specific gappenalties and locally reduced gappenalties inhydrophilic regions encourage newgapsinpotential loopregions rather thanregular secondary structure. Fourthly, positions inearly alignments wheregapshavebeenopened receive locally reduced gappenalties toencourage the opening upofnewgapsatthesepositions. These modifications areincorporated intoanewprogram, CLUSTALW whichisfreely available.
Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and polycyclic phenylphenalenones), but no such activity has been reported. Root cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable source to search for such enzymes because they synthesize large amounts of phenylphenalenones, but no other products that are known to require CHSs or related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy led to the identification of cDNAs for a type III PKS sharing only approximately 60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora polyketide synthase 1). The purified recombinant protein accepted a large variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl- and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA as starter and a single condensation reaction to a diketide. Benzalacetones, the expected release products, were observed only with unsaturated phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA (80% of the products). With all other substrates, WtPKS1 performed two condensation reactions and released pyrones. We propose that WtPKS1 catalyses the first step in diarylheptanoid biosynthesis and that the observed pyrones are derailment products in the absence of downstream processing proteins.
An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate
and easy to use on microcomputers. The approach is based on the conventional dynamic-programming method of pairwise alignment.
Initially, a hierarchical clustering of the sequences is performed using the matrix of the pairwise alignment scores. The
closest sequences are aligned creating groups of aligned sequences. Then close groups are aligned until all sequences are
aligned in one group. The pairwise alignments included in the multiple alignment form a new matrix that is used to produce
a hierarchical clustering. If it is different from the first one, iteration of the process can be performed. The method is
illustrated by an example : a global alignment of 39 sequences of cytochrome c.
Chalcone synthase (CHS) catalyzes the first and key regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. In bean (Phaseolus vulgaris L.) and other members of the Leguminoseae, chalcone synthase is also involved in the synthesis of the isoflavonoid-derived phytoalexin antibiotics characteristic of this family. We have demonstrated that the haploid genome of bean contains a family of about six to eight CHS genes, some of which are tightly clustered. Treatment of bean cells with fungal elicitor activates several of these genes leading to the accumulation of at least five and probably as many as nine distinct CHS transcripts encoding a set of CHS isopolypeptides of Mr 42-43 kDa but with differing pI in the range pH 6-7. In elicited cells specific transcripts and encoded polypeptides are differentially induced with respect to both the extent and kinetics of accumulation. Wounding or infection of hypocotyl tissue also activates several CHS genes with marked differences in the pattern of accumulation of specific transcripts and encoded polypeptides in wounded compared to infected tissue or elicited cells, indicating operation of more than one cue for defense gene activation. Illumination induces accumulation of a different set of CHS transcripts including only one of the set hitherto demonstrated to be induced by biological stress. The organization and differential regulation of the CHS gene family in bean are discussed in relation to the functions of this enzyme in adaptative and protective responses to diverse environmental stresses.
[6]-Gingerol, a major pungent ingredient found in the rhizome of ginger, has been reported to possess a strong antiinflammatory activity, which is considered to be closely associated with its cancer chemopreventive potential. [6]-Paradol, another pungent phenolic substance found in ginger and other Zingiberaceae plants, also has a vanilloid structure found in other chemopreventive phytochemicals including curcumin. In the present study, [6]-gingerol and [6]-paradol were found to exert inhibitory effects on the viability and DNA synthesis of human promyelocytic leukemia (HL-60) cells. The cytotoxic and antiproliferative effects of both compounds were associated with apoptotic cell death. The above results suggest that [6]-gingerol and [6]-paradol possess potential cytotoxic/cytostatic activities.
Benzalacetone synthase (BSA) is a novel plant-specific polyketide synthase that catalyzes a one step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 skeleton of phenylbutanoids in higher plants. A cDNA encoding BAS was for the first time cloned and sequenced from rhubarb (Rheum palmatum), a medicinal plant rich in phenylbutanoids including pharmaceutically important phenylbutanone glucoside, lindleyin. The cDNA encoded a 42-kDa protein that shares 60-75% amino-acid sequence identity with other members of the CHS-superfamily enzymes. Interestingly, R. palmatum BAS lacks the active-site Phe215 residue (numbering in CHS) which has been proposed to help orient substrates and intermediates during the sequential condensation of 4-coumaroyl-CoA with malonyl-CoA in CHS. On the other hand, the catalytic cysteine-histidine dyad (Cys164-His303) in CHS is well conserved in BAS. A recombinant enzyme expressed in Escherichia coli efficiently afforded benzalacetone as a single product from 4-coumaroyl-CoA and malonyl-CoA. Further, in contrast with CHS that showed broad substrate specificity toward aliphatic CoA esters, BAS did not accept hexanoyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and acetyl-CoA as a substrate. Finally, besides the phenylbutanones in rhubarb, BAS has been proposed to play a crucial role for the construction of the C6-C4 moiety of a variety of natural products such as medicinally important gingerols in ginger plant.
A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis Induction of apoptosis in HL-60 cells by pungent vaniloids, [6]-gingerol and [6]-paradol
- S Brand
- D Holscher
- A Schierborn
- A Svatos
- J Schroder
- Schneider
- E Lee
- Surh
Brand S, Holscher D, Schierborn A, Svatos A, Schroder J , Schneider B (2006) .A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis, Planta. 224: 413-28.1. Lee E, Surh YJ (1998) Induction of apoptosis in HL-60 cells by pungent vaniloids, [6]-gingerol and [6]-paradol. Cancer Lett. 134 ,163-168