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.     

Domain structure and interaction within the pentafunctional arom polypeptide.

The AROM locus of Aspergillus nidulans specifies a pentafunctional polypeptide catalysing five consecutive steps leading to the production of 5-enolpyruvylshikimate 3-phosphate in the shikimate pathway. Aided by oligonucleotide-mediated site-directed mutagenesis, the whole AROM locus and various overlapping subfragments from within it have been fused to the powerful hybrid trc promoter in the Escherichia coli plasmid pKK233-2. Expression of these subfragments in appropriate aro mutants of E. coli has (a) allowed the delineation of functional domains within the arom polypeptide, (b) shown that the arom polypeptide falls in two independently folding and functioning regions, the N-terminal half specifying 3-dehydroquinate (DHQ) synthase and EPSP synthase and the C-terminus specifying shikimate kinase, biosynthetic 3-dehydroquinase (DHQase) and shikimate dehydrogenase, and (c) strongly suggested an interaction between the DHQ synthase and EPSP synthase domains to stabilise the EPSP synthase activity. In addition an isoenzyme of biosynthetic DHQase, catabolic DHQase, encoded by the QUTE gene of A. nidulans has been transcribed from the trc promoter and upon isopropyl-thio-beta-D-galactoside induction produces up to 20% of the total soluble cell protein.     

Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.Abbreviations EPSP 5-enolpyruvylshikimic acid 3-phosphate     

In vivo overproduction of the pentafunctional arom polypeptide in Aspergillus nidulans affects metabolic flux in the quinate pathway

Summary The shikimate pathway and the quinic acid utilisation (QUT) pathway of Aspergillus nidulans and other fungi share the two common metabolic intermediates, 3-dehydroquinic acid (DHQ) and dehydroshikimic acid (DHS), which are interconverted by two isoenzymes, catabolic 3-dehydroquinase, (cDHQase) and biosynthetic dehydroquinase (bDHQase). bDHQase is one of five consecutive enzymatic activities associated with the pentafunctional arom protein encoded by the complex AROM locus, whereas cDHQase is encoded by the single-function QUTE gene, one of seven genes comprising the QUT gene cluster in A. nidulans, which is required for the catabolism of quinate to protocatechuate. We addressed the question of how much (if any) leakage there is of the two common substrates between the two pathways, by increasing the concentration of the arom protein in vivo by means of recombinant DNA technology. We demonstrated that constitutive overproduction of the arom protein by 12-fold in the presence of quinate inhibits germination of conidiospores, but showed that 12-fold quinate-inducible overproduction of arom protein does not have this effect. In addition we showed that a qutE mutant (lacking cDHQase) can grow with quinic acid as sole carbon source when the arom protein is overproduced fivefold. The data are most simply interpreted as simple competition for common substrates by the enzymes of the two pathways and demonstrate that any channelling function of the arom protein in vivo is relatively leaky.     

Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate

Cultured cells of the higher plant Corydalis sempervirens Pers. which had been adapted to growing in the presence of 5 mM glyphosate (N-[phosphonomethyl]-glycine), a herbicide and a potent specific inhibitor of the shikimate pathway enzyme 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, had a nearly 40-fold increased level of the extractable activity of EPSP synthase. Activities of five other shikimate pathway enzymes were, however, similar in the adapted and nonadapted cells, and the concentrations of the free aromatic amino acids in the two cell lines were also similar. EPSP synthases purified from glyphosate-adapted, as well as nonadapted cells, had identical physical, kinetic, and immunological properties, which indicated that the glyphosate-sensitive enzyme was overproduced in the adapted culture. Overproduction of EPSP synthase in the adapted culture was unequivocally established by two-dimensional polyacrylamide gel electrophoresis, as well as by one-dimensional sodium dodecyl sulfate-gradient gel electrophoresis and quantitation of EPSP protein by immunoassay after transfer to nitrocellulose membranes. While about 0.06% of the total soluble protein from nonadapted cells was EPSP synthase protein, the proportion was 2.6% in the adapted cells. In vivo pulse-labeling experiments with [35S]methionine established that the adapted cells have an increased rate of EPSP synthase protein synthesis.     

Purification and Properties of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Dark-Grown Seedlings of Sorghum bicolor

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent K_m values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (K_i = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.     

Aggregation and separability of the shikimate pathway enzymes in yeasts

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.     

核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶的酶学性质

核盘菌5-烯醇丙酮酰莽草酸-3-磷酸合酶（EPSP合酶）是AROM多功能酶的活性之一.该酶催化莽草酸磷酸（S3P）和磷酸烯醇式丙酮酸（PEP）产生5-烯醇丙酮酰莽草酸-3-磷酸和无机磷酸的可逆反应,受除草剂草甘膦（N-（膦羧甲基）甘氨酸）抑制.纯化了核盘菌AROM蛋白并对EPSP合酶进行了酶学特征研究.结果显示,该酶反应的最适pH值为7.2,最适温度为30℃.热失活反应活化能是69.62 kJ/mol.底物S3P和PEP浓度分别高于1 mmol/L和2 mmol/L时,对EPSP合酶反应产生抑制作用.用双底物反应恒态动力学Dalziel方程求得的Km(PEP)为140.98 μmol/L,K _m(S3P)为139.58 μmol/L.酶动力学模型遵循顺序反应机制.草甘膦是该酶反应底物PEP的竞争性抑制剂（Ki为0.32 μmol/L）和S3P的非竞争性抑制剂.正向反应受K⁺激活.当［K⁺］增加时,K _m(PEP)随之降低,Km(S3P)不规律变化,而K _i(PEP)随［K⁺］增加而提高.     

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     

Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway

The bifunctional enzyme dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroshikimate and the reduction of dehydroshikimate to shikimate in the shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the shikimate pathway is achieved by increasing the effective concentration of dehydroshikimate through the proximity of the two sites.     

Genome mining of mycosporine-like amino acid (MAA) synthesizing and non-synthesizing cyanobacteria: A bioinformatics study

Mycosporine-like amino acids (MAAs) are a family of more than 20 compounds having absorption maxima between 310 and 362 nm. These compounds are well known for their UV-absorbing/screening role in various organisms and seem to have evolutionary significance. In the present investigation we tested four cyanobacteria, e.g., Anabaena variabilis PCC 7937, Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301, for their ability to synthesize MAA and conducted genomic and phylogenetic analysis to identify the possible set of genes that might be involved in the biosynthesis of these compounds. Out of the four investigated species, only A. variabilis PCC 7937 was able to synthesize MAA. Genome mining identified a combination of genes, YP_324358 (predicted DHQ synthase) and YP_324357 (O-methyltransferase), which were present only in A. variabilis PCC 7937 and missing in the other studied cyanobacteria. Phylogenetic analysis revealed that these two genes are transferred from a cyanobacterial donor to dinoflagellates and finally to metazoa by a lateral gene transfer event. All other cyanobacteria, which have these two genes, also had another copy of the DHQ synthase gene. The predicted protein structure for YP_324358 also suggested that this product is different from the chemically characterized DHQ synthase of Aspergillus nidulans contrary to the YP_324879, which was predicted to be similar to the DHQ synthase. The present study provides a first insight into the genes of cyanobacteria involved in MAA biosynthesis and thus widens the field of research for molecular, bioinformatics and phylogenetic analysis of these evolutionary and industrially important compounds. Based on the results we propose that YP_324358 and YP_324357 gene products are involved in the biosynthesis of the common core (deoxygadusol) of all MAAs.     

Recombinant Escherichia coli Strain Produces a ZZ Domain Displaying Biopolyester Granules Suitable for Immunoglobulin G Purification

The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could be enhanced by the overproduction of the ZZ-PHA synthase. ZZ-PHA granules enabled efficient purification of IgG from human serum.     

Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line

Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 mM glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogeneous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with Mr 49,000-55,800. Km values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 18 microM, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate (Ki = 0.17 microM). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.     

Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.     

Identification of two new genes,mukE andmukF,involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) has received attention in the past because it is the target of the broad-spectrum herbicide glyphosate. The natural substrate of EPSP synthase is shikimate-3-phosphate. However, this enzyme can also utilize shikimate as substrate. Remarkably, this reaction is insensitive to inhibition by glyphosate. Crystallographic analysis of EPSP synthase from Escherichia coli, in complex with shikimate/glyphosate at 1.5 Angstroms resolution, revealed that binding of shikimate induces changes around the backbone of the active site, which in turn impact the efficient binding of glyphosate. The implications from these findings with respect to the design of novel glyphosate-insensitive EPSP synthase enzymes are discussed.     

Substitution of Gly-96 to Ala in the 5-enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae results in a greatly reduced affinity for the herbicide glyphosate

The aroA gene of Klebsiella pneumoniae encoding the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, which is the target of the herbicide glyphosate, was cloned and sequenced from both the wild-type and the glyphosate-resistant mutant K. pneumoniae K1, which possesses a glyphosate-insensitive EPSP synthase. Both genes were expressed in Escherichia coli and were capable of complementing an auxotrophic aroA mutation. The transformed cells showed increased tolerance to glyphosate due to the overproduction of either the mutant or the wild type EPSP synthase. Nucleotide sequence analysis of the K. pneumoniae aroA gene indicated a protein-coding region of 427 amino acids with a derived Mr for the EPSP synthase of 45,976. Comparison of the two aroA alleles showed a single base change resulting in a substitution of Gly-96 to Ala in the deduced amino acid sequence. By comparison with other known EPSP synthase sequences the mutation was shown to be located in a highly conserved region, indicating that this region is essential for the binding of the herbicide glyphosate.     

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.     

Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W₁₀BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.