Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Molecular characterization and expression analysis on two isogenes encoding 3-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-<Emphasis Type="Italic">arabino</Emphasis>-heptulosonate 7-phosphate synthase in grapes

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) is an entry enzyme of the shikimate pathway that connects primary carbohydrate metabolism with the biosynthesis of most secondary metabolites in plants. In the present study, two DAHPS cDNAs were cloned from grape berries (Vitis vinifera) and designated as VvDAHPS-1 and VvDAHPS-2. These two cDNA sequences share 75.7% of the identities. Their DNA corresponding to the two isogenes both contain four introns. The deduced proteins from two cDNAs had different NH₄-terminal regions and putative mature regions sharing sequence, molecular size and pI value similarity. Both of VvDAHPSs had a close evolution relationship with Populus trichocarpa DAHPSs. The prokaryotically-expressed VvDAHPSs both manifested DAHPS catalytic activity and Mn²⁺-activated effects. Analysis by real time-PCR showed that VvDAHPS-1 and VvDAHPS-2 were expressed in all the tested tissues, but their expression patterns accompanying with berry mature varied in the skin, pulp and seeds. The results give new insight into further study on regulatory mechanism of grape phenolics biosynthesis.     

利用酵母双杂交系统筛选水稻类受体激酶OsWAK50胞内域相互作用蛋白

细胞壁连接的类受体激酶(wall-associated kinase,WAK)是植物细胞中一类特有的类受体激酶基因亚家族,因其胞外域与细胞壁紧密相连而得名．水稻中共有125个OsWAK基因,OsWAK50编码的蛋白质具有胞外域、跨膜域和激酶域,呈现典型的WAK样受体激酶特征．首先通过对OsWAK50-GFP融合蛋白的观察发现OsWAK50定位于细胞膜并且与细胞壁偶联．进而通过酵母双杂交系统筛选到了20个可能与OsWAK50胞内域相互作用的候选蛋白,并通过一对一酵母转化验证了OsSK4、OsSWIB和OsSWI3C全长均可与OsWAK50胞内域相互作用．进一步分析显示,OsSWIB能够直接与OsWAK50激酶域互作,而OsSK4和OsSWI3C与OsWAK50胞内域的互作是依赖于OsWAK50 C端的．研究还表明,OsSK4和OsSWIB亦能与OsWAK50同源基因OsWAK53a结合,而OsSWI3C则不能与OsWAK53a结合．双分子荧光互补实验证明,OsSK4与OsWAK50和OsWAK53a能够在植物体内发生互作．以上结果为阐明OsWAK50发挥功能的分子机制提供了重要线索．     

Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICB^Glc, and then captured by β-glucosidase.     

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase is regulated for the accumulation of polysaccharide-linked hydroxycinnamoyl esters in rice (Oryza sativa L.) internode cell walls

Polysaccharide-linked hydroxycinnamoyl esters (PHEs) over-accumulate in the internodes of a rice (Oryza sativa L.) mutant, Fukei 71 (F71). This accumulation is accompanied by over-expression of phenylalanine ammonialyase (PAL). In this study, we show that only one member of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) family expresses in close correlation with PAL. Furthermore, substrate availability to DAHPS is promoted by down-regulating the expression of plastidic pyruvate kinase (PKp), a competitor of DAHPS. Since the over-production of PHEs is caused by D50 gene disruption, these results suggest that specific enzymes in the phenylpropanoid and shikimate pathways are coordinately up-regulated. In addition, the results indicate that carbon-flow into the shikimate pathway is modified for the synthesis of PHEs, and is probably controlled by D50.     

A spectrum of genes expressed during early stages of rice panicle and flower development

To unravel gene expression patterns during rice inflorescence development, particularly at early stages of panicle and floral organ specification, we have characterized random cloned cDNAs from developmental-stage-specific libraries. cDNA libraries were constructed from rice panicles at the stage of branching and flower primordia specification or from panicles undergoing floral organogenesis. Partial sequence analysis and expression patterns of some of these random cDNA clones from these two rice panicle libraries are presented. Sequence comparisons with known DNA sequences in databases reveal that approximately sixtyeight per cent of these expressed rice genes show varying degrees of similarity to genes in other species with assigned functions. In contrast, thirtytwo per cent represent uncharacterized genes. cDNAs reported here code for potential rice homologues of housekeeping molecules, regulators of gene expression, and signal transduction molecules. They comprise both single-copy and multicopy genes, and genes expressed differentially, both spatially and temporally, during rice plant development. New rice cDNAs requiring specific mention are those with similarity toCOP1, a regulator of photomorphogenesis inArabidopsis; sequence-specific DNA binding plant proteins like AP2-domain-containing factors; genes that specify positional information in shoot meristems like leucine-rich-repeat-containing receptor kinases; regulators of chromatin structure like Polycomb domain protein; and also proteins induced by abiotic stresses.     

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH₂-terminal deletion constructs of P70R. Among the NH₂-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH₂-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH₂-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH₂-terminal hydrophobic TMS1. Furthermore, attachment of the NH₂-terminal hydrophilic domain to the NH₂-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH₂-terminal region determines the subcellular localization of ABC subfamily D proteins.     

Mode of action of glyphosate in Candida maltosa

The broad-spectrum herbicide glyphosate inhibits the growth of Candida maltosa and causes the accumulation of shikimic acid and shikimate-3-phosphate. Glyphosate is a potent inhibitor of three enzymes of aromatic amino acid biosynthesis in this yeast. In relation to tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and dehydroquinate synthase, the inhibitory effect appears at concentrations in the mM range, but 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase is inhibited by micromolar concentrations of glyphosate. Inhibition of partially purified EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate (PEP) with a K _i -value of 12 M. The app. K _m for PEP is about 5-fold higher and was 62 M. Furthermore, the presence of glyphosate leads to derepression of many amino acid biosynthetic enzymes.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - EPSP synthase 5-enolpyruvylshikimate 3-phosphate synthase - PEP phosphoenolpyruvate - S-3-P shikimate-3-phosphate     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Purification and properties of a glyphosate-tolerant 5-enolpyruvylshikimate 3-phosphate synthase from the cyanobacterium Anabaena variabilis

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.9) from the glyphosate-tolerant cyanobacterium Anabaena variabilis (ATCC 29413) was purified to homogeneity. The enzyme had a similar relative molecular mass to other EPSP synthases and showed similar kinetic properties except for a greatly elevated K _i for the herbicide glyphosate (approximately ten times higher than that of enzymes from other sources). With whole cells, the monoisopropylamine salt of glyphosate was more toxic than the free acid but the effects of the free acid and monoisopropylamine salt on purified EPSP synthase were identical.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate - M_r relative molecular mass - PEP phosphoenolpyruvate - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - S3P shikimate 3-phosphate The funding of this work by the Agricultural and Food Research Council and the University of Dundee Research Initiatives Programme is gratefully acknowledged.     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data

Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides.     

Evaluation of the toxicity of stress-related aldehydes to photosynthesis in chloroplasts

Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO₂ photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. α,β-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO₂-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H₂O → ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.     

Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction

Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141 g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48 h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.