Pyrone polyketides synthesized by a type III polyketide synthase from Drosophyllum lusitanicum

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.     

5-enolpyruvylshikimate 3-phosphate synthase, the target enzyme of the herbicide glyphosate, is synthesized as a precursor in a higher plant

Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30- to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M_r) 53900 ± 900 as compared to M_r 45500 ± 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures.     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Probing biosynthesis of plant polyketides with synthetic N-acetylcysteamine thioesters

Recombinant chalcone synthase (CHS) from Scutellaria baicalensis accepted cinnamoyl diketide-NAC and cinnamoyl-NAC as a substrate, and carried out sequential condensations with malonyl-CoA to produce 2',4',6'-trihydroxychalcone. Steady-state kinetic analysis revealed that the CHS accepted the diketide-NAC with less efficiency, while cinnamoyl-NAC primed the enzyme reaction almost as efficiently as cinnamoyl-CoA. On the other hand, it was for the first time demonstrated that the diketide-NAC was also a substrate for recombinant polyketide reductase (PKR) from Glycyrrhiza echinata, and converted to the corresponding beta-ketohemithioester. Furthermore, by co-action of the CHS and the PKR, the NAC-thioesters were converted to 6'-deoxychalcone in the presence of NADPH and malonyl-CoA.     

Biosynthesis of polyketides. Purification and inhibition studies of 6-methylsalicylic acid synthase

6-MSA³ synthase has been purified 190-fold with 33% yield. The purification was found to be dependent on the presence of glycerol. The acetylenic inhibitors 3-pentynoyl- and 2-hexynoyl-NAC completely inhibit 6-MSA production at concentrations in which fatty acid synthesis, TAL production as well as NADPH oxidation are only partially affected. These results confirm earlier studies on the specificity of inhibition by acetylenic inhibitors and support a mechanism wherein the NADPH-mediated reduction step occurs on a 6-carbon rather than on an 8-carbon intermediate.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Localization of stilbene synthase in Vitis vinifera L. during berry development

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.     

Characterization of a pine multigene family containing elicitor- responsive stilbene synthase genes

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial -glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.     

A single change of histidine to glutamine alters the substrate preference of a stilbene synthase.

Stilbene and chalcone synthases are related polyketide synthases which use the same substrates but form different products. The environment of the condensing active site cysteine is highly conserved, except for the positions -2 and -3. All chalcone synthases contain Gln-Gln and prefer 4-coumaroyl-CoA as starter CoA ester, while the two known stilbene synthases contain Gln-His or His-Gln (preference phenylpropionyl-CoA and 4-coumaroyl-CoA, respectively). We investigated whether the presence and/or position of the histidine influences the substrate preference and the product specificity (stilbene or chalcone). The two amino acid motifs in the chalcone synthase from Pinus sylvestris (Gln-Gln) and in the stilbene synthases from P. sylvestris (Gln-His) and Arachis hypogaea (His-Gln) were changed by site-directed mutagenesis into all sequence combinations as found in the natural enzymes. Assays with the mutant proteins showed that the histidine does not determine the product specificity. With the chalcone and the stilbene synthase from P. sylvestris, any sequence deviation reduced the activity without marked effects on the substrate preference. The stilbene synthase from A. hypogaea was different. The change from His-Gln to Gln-His abolished enzyme activity almost completely with all three substrates. The change to Gln-Gln selectively reduced the activity with 4-coumaroyl-CoA, and the kinetic analysis indicated a slight increase in Km and a 3-fold reduction of Vmax, when compared with the parent enzyme. This converted the enzyme from a resveratrol-forming into a dihydropinosylvin-forming stilbene synthase.     

Molecular cloning and analysis of a cDNA coding for chorismate synthase from the higher plant Corydalis sempervirens Pers.

Chorismate synthase catalyzes the last common step in the biosynthesis of the three aromatic amino acids in microorganisms and plants. We have cloned a cDNA for this enzyme from the higher plant Corydalis sempervirens. This is the first chorismate synthase cDNA from a eukaryotic organism. The nucleotide sequence was determined and the identity of the cDNA was confirmed by the amino acid sequence of tryptic peptides obtained from purified chorismate synthase. The homology to the two known bacterial sequences is about 48%. The cDNA contains an open reading frame of 1341 base pairs, encoding a protein of 447 amino acids. This protein with a molecular mass of 48,100 daltons resembles a chorismate synthase precursor targeted for chloroplast import. Multiple sites of polyadenylation were observed in chorismate synthase mRNAs.     

Reconstitution of intermediate filaments from a higher plant.

Immunological studies have shown that plants contain intermediate-filament antigens, but it is not known whether these proteins are capable in themselves of forming filaments. To address this problem, a detergent-resistant and high-salt-insoluble fraction from carrot (Daucus carota L.) suspension cells was solubilized with 9 M-urea and then subjected to a two-step dialysis procedure, devised for the reconstitution of animal intermediate filaments. This induced the self-assembly of 10 nm filaments and large bundles of filaments. The predominant components of reconstituted material were polypeptides with apparent molecular masses between 58 and 62 kDa. These polypeptides immunoblotted with two monoclonal antibodies known to show broad cross-reactivity with intermediate filaments across the phylogenetic spectrum. This establishes that the antigens are able to self-assemble into intermediate-sized filaments.     

beta-Furfuryl-beta-glucoside. An endogenous activator of higher plant UDP-glucose: (1----3)-beta-glucan synthase.

We have recently established the existence of endogenous activators of higher plant UDP-glucose: (1----3)-beta-glucan synthase (Callaghan, T., Ross, P., Weinberger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1099-1103). Here we report the purification and chemical analysis of the most abundant and specific compound, termed Activator I, isolated from Vigna radiata. This compound was extensively purified by a multistep procedure which yielded 0.1 mg of purified activator/g of fresh tissue. Enzyme digestion, neutral sugar analysis, GC/MS of permethylated derivatives, and NMR analysis of native Activator I indicated that the compound contains a single beta-linked glucosyl residue. High resolution FAB-MS indicated an elemental composition of C11H16O7 (Mr = 260), with a calculated Mr of 98 for the aglycone. 13C, DEPT, and COSY NMR spectra showed that the aglycone molecule is an oxygen heterocycle of 5 carbons, consistent with a structure of beta-furfuryl alcohol. Comparison of IR and GC/EI-MS spectra of authentic beta-furfuryl alcohol with native aglycone confirmed the conclusion that Activator I is beta-furfuryl-beta-glucoside. Chemically synthesized beta-furfuryl-beta-glucoside has identical chemical properties and biological activity when compared with the purified endogenous activator (Ka = 50 microM).     

Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase.

Addition of arachidonic acid or 5-phenyl-4-pentenylhydroperoxide to manganese-prostaglandin endoperoxide synthase (Mn-PGH synthase) produced a species with an absorbance maximum at 418 nm. This maximum is distinct from those of resting enzyme (372 and 468 nm) or reduced enzyme (434 nm). The formation of the 418 nm-absorbing species was observed immediately after the addition of hydroperoxide to enzyme but only after a 10-s lag period following addition of arachidonate. Mn-PGH synthase exhibited a peroxidase activity that was 0.8% that of Fe-PGH synthase. Addition of peroxidase reducing substrates to the oxidized form of Mn-PGH synthase diminished the absorbance at 418 nm. In the case of N,N,N',N'-tetramethylphenylenediamine, reduction of the 418 nm-absorbing species was accompanied by an increase in absorbance at 610 nm due to the oxidized form of the amine. Thus, the spectral and chemical properties of the 418 nm-absorbing species are consistent with its existence as a higher oxidation state of Mn-PGH synthase. Kinetic analysis indicated that formation of the higher oxidation state preceded or was coincident with oxygenation of the fatty acid substrate, eicosa-11,14-dienoic acid. The cyclooxygenase activity of Mn-PGH synthase was inhibited by the combination of glutathione and human plasma glutathione peroxidase at a glutathione peroxidase concentration 227-fold lower than the concentration that inhibited Fe-PGH synthase. The results suggest that Mn-PGH synthase forms a higher oxidation state following reaction with hydroperoxides added exogenously or generated endogenously from polyunsaturated fatty acid substrates. This higher oxidation state functions in the peroxidase catalytic cycle of Mn-PGH synthase, and its formation appears to be essential for activation of the cyclooxygenase catalytic cycle.     

Constitutive accumulation of cis-piceid in transgenic Arabidopsis overexpressing a sorghum stilbene synthase gene

Sorghum SbSTS1 was the first example of a stilbene synthase gene in monocots. Previously, we demonstrated that the gene was involved in defense responses. To examine its biochemical function in planta, SbSTS1 was overexpressed in transgenic Arabidopsis. Metabolite analysis revealed that cis-resveratrol glucoside (piceid) accumulated as the major stilbene in the transgenic lines. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode, up to 580 microg g(-1) FW of cis-piceid were detected in 2-week-old plants, which represent a convenient source of the cis-isomers for pharmacological investigations. Our results also suggested the presence of unknown stilbene isomerase activities in Arabidopsis.     

Novel secA alleles improve export of maltose-binding protein synthesized with a defective signal peptide.

Mutations previously designated prlD were described that suppressed malE signal sequence mutations and were located in the vicinity of the secA gene on the Escherichia coli chromosome. In this study, we demonstrated that four such independently isolated prlD mutations represented three unique single-base substitutions in secA, resulting in alterations at residues 111, 373, and 488 of the 901-residue SecA protein. Heretofore, the only mutations that had been described for secA were located early in the gene and resulted in a general protein export defect. Insertion mutations in the cloned gene X-secA operon that reduced or eliminated suppression by a prlD mutation also have been obtained. The properties of these suppressor and insertion mutations provide some insight into the role of SecA in the protein export process.     

Localization of a centrin-like protein to higher plant plasmodesmata.

Antibodies against centrin, the ubiquitous calcium-binding contractile protein, recognized a 17 kDa protein in extracts of onion root tips and cauliflower florets. Using immunofluorescence microscopy, anti-centrin antibodies were localized to the developing cell plate of onion and cauliflower root tip cells. In cauliflower florets, these antibodies localized to the walls in a punctate manner, consistent with the distribution of plasmodesmata as shown by colocalization with callose. Anti-centrin antibodies were localized to plasmodesmata of onion root tips and cauliflower florets with immunogold electron microscopy. Furthermore, this label was concentrated around the necks of plasmodesmata. In contrast, an antibody against calmodulin, which is a closely related calcium-binding protein, did not label plasmodesmata. We propose that centrin is a component of calcium-sensitive contractile nanofilaments in the neck region of plasmodesmata and facilitates the calcium-induced regulation of intercellular transport.     

Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut

Stilbene synthase ( resveratrol -forming) converts one molecule of rho- coumaroyl -CoA and three molecules of malonyl-CoA into 3,4',5- trihydroxystilbene . Following selective induction of stilbene synthesis in cell suspension cultures of peanut (Arachis hypogaea), the enzyme was extracted and purified to apparent homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. The enzyme was found to be a dimer of estimated Mr = 90,000 exhibiting under denaturing conditions a subunit Mr of approximately 45,000. The isoelectric point was determined with pI = 4.8. The enzyme's high selectivity towards rho- coumaroyl -CoA (Km = 2 microM) as substrate qualified it as resveratrol -forming stilbene synthase. Structurally related CoA esters, e.g. dihydro-rho- coumaroyl -CoA and cinnamoyl-CoA, were converted less than 1/10 as efficiently as rho- coumaroyl -CoA. Malonyl-CoA (Km = 10 microM) could not be substituted by acetyl-CoA. The purified enzyme was free of chalcone synthase activity. Antibodies raised against stilbene synthase were shown to be monospecific and not to cross-react with chalcone synthase.