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The anti-tuberculosis (TB) agent IMB-T130 was speculated to be a multi-target compound. In this research, we found that IMB-T130 inhibits the catalytic activity of Mycobacterium tuberculosis 3-dehydroquinate synthase (MtDHQS), the enzyme in the second step of the shikimate pathway. IMB-T130 was identified as a selective inhibitor of MtDHQS with an IC50 value of 0.87 μg/mL. The interaction between the compound and protein was analysed by surface plasmon resonance and circular dichroism. Based on the in silico molecular docking results, the essential amino acids in the binding pocket were then confirmed by site-directed mutagenesis. Overexpression of DHQS reduced the antibacterial activity of IMB-T130 in cells, verifying that DHQS is the target of IMB-T130. IMB-T130 inhibited standard and drug-resistant M. tuberculosis strains by targeting DHQS. Our findings improve our understanding of MtDHQS and make it to be a potential target for new anti-TB drug discovery.

The shikimate pathway is as an attractive target because it is present in bacteria, algae, fungi, and plants but does not occur in mammals. In Mycobacterium tuberculosis (MTB), the shikimate pathway is integral to the biosynthesis of naphthoquinones, menaquinones, and mycobactin. In these study, novel inhibitors of 3-dehydroquinate synthase (DHQS), an enzyme that catalyzes the second step of the shikimate pathway in MTB, were determined. 12,165 compounds were selected from two public databases through virtual screening and molecular docking analysis using PyRx 8.0 and Autodock 4.2, respectively. A total of 18 compounds with the best binding energies (-13.23 to -8.22 kcal/mol) were then selected and screened for absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis, and nine of those compounds were found to satisfy all of the ADME and toxicity criteria. Among those nine, the three compounds-ZINC633887 (binding energy = -10.29 kcal/mol), ZINC08983432 (-9.34 kcal/mol), and PubChem73393 (-8.61 kcal/mol)-with the best binding energies were further selected for molecular dynamics (MD) simulation analysis. The results of the 50-ns MD simulations showed that the two compounds ZINC633887 and PubChem73393 formed stable complexes with DHQS and that the structures of those two ligands remained largely unchanged at the ligand-binding site during the simulations. These two compounds identified through docking and MD simulation are potential candidates for the treatment of TB, and should undergo validation in vivo and in vitro.

Bagremycin A and B, produced by Streptomyces sp. Tu 4128, are two antibiotics active against Gram-positive bacteria and fungi. To elucidate the biosynthetic pathway of bagremycins, two genes named bagB and bagC, were cloned and characterized from a bagremycin-producing strain. The deduced protein products of bagB and bagC show high sequence identity with aldolase and 3-dehydroquinate synthase, respectively. The bagC gene is located immediately downstream of the bagB gene and subsequent reverse transcription and polymerase chain reaction analysis revealed that bagB is co-transcribed with bagC. Inactivation of either bagB or bagC resulted in the complete abolishment of bagremycin production in fermented cultures, which suggests that these two genes are involved in the biosynthesis of bagremycins. The results from this study will allow acquisition of the entire biosynthetic cluster for bagremycins and further our understanding of the biosynthetic pathway and mechanism of action of bagremycin.

The disease bacterial blight results in serious production losses of rice in Asian countries. The aroB gene encoding dehydroquinate synthase (DHQS), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo). DHQS plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. The aroB gene (Xoo1243) was cloned from Xoo and the corresponding DHQS protein was subsequently overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method and yielded crystals that diffracted to 2.5 A resolution. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 118.2, c = 98.2 A. According to a Matthews coefficient calculation, the crystal contained two molecules in the asymmetric unit, with a corresponding V(M) of 2.06 A(3) Da(-1) and a solvent content of 40.4%.

The aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis, is responsible for millions of deaths annually. The increasing prevalence of the disease, the emergence of multidrug-resistant strains, and the devastating effect of human immunodeficiency virus co-infection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. Since the shikimate pathway is present and essential in algae, higher plants, bacteria, and fungi, but absent from mammals, the gene products of the common pathway might represent attractive targets for the development of new antimycobacterial agents. In this review we describe studies on shikimate pathway enzymes, including enzyme kinetics and structural data. We have focused on mycobacterial shikimate pathway enzymes as potential targets for the development of new anti-TB agents.

The role of the active site metal in determining binding to 3-dehydroquinate synthase has been examined. Protocatechuic acid, catechol, and derivatives of these aromatics were synthesized that shared the common element of an ortho dihydroxylated benzene ring. Inhibition constants were determined for each aromatic as well as the variation of this inhibition as a function of whether Co+2 or Zn+2 was the active site metal ion.

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.Key words: Corynebacterium glutamicum, aromatic amino acid biosynthetic gene, dehydroquinate synthase, aroB gene.

The aroB gene of Salmonella typhimurium, encoding dehydroquinate synthase, has been cloned into pUC19 and the DNA sequence determined. The aroB gene was isolated from a cosmid gene bank by complementation of an Escherichia coli aroB mutant and screening by Southern blot analysis. The nucleotide sequence of the S. typhimurium aroB gene revealed the presence of an open reading frame, encoding a protein of 362 amino acids with a calculated molecular mass of 38696 Daltons. The amino acid sequence of S. typhimurium dehydroquinate synthase is nearly identical to the E. coli homologue and shows high homology with other aroB gene products from other organisms. Subsequently, a stable insertional mutation in aroB was introduced into the wild-type S. typhimurium C5 strain. This mutant was auxotrophic for aromatic compounds. Infection of BALB/c mice with this mutant demonstrated attenuation comparable to other S. typhimurium mutants unable to biosynthesize aromatic amino acids.

Cyclohexenyl and cyclohexylidene inhibitors possessing strategically placed olefinic residues, in general, bind to 3-dehydroquinate (DHQ) synthase more tightly than similarly substituted cyclohexyl inhibitors. All of the newly synthesized inhibitors were prepared from a common DHQ derivative. Cyclohexenyl phosphate 1 is the most potent inhibitor of DHQ synthase thus far identified with an inhibition constant (Ki = 1.2 × 10-10 M), indicating active site binding 1000-fold tighter relative to the corresponding cyclohexyl phosphate 5. Cyclohexenyl tricarboxylate 2 binds 700-fold more tightly than similarly substituted cyclohexyl tricarboxylate 6 and is the first example of a nanomolar-level inhibitor (Ki = 8.6 × 10-9 M) possessing neither a phosphate monoester nor a phosphonic acid. Cyclohexenyl homophosphonate 4 (Ki = 3.0 × 10-8 M) and cyclohexylidene homophosphonate 10 (Ki = 3.2 × 10-9 M) bind 57- and 530-fold, respectively, more tightly than the corresponding cyclohexyl homophosphonate 8. Cyclohexylidene h...

Crystallization of Aspergillus nidulans 3-dehydroquinate synthase (DHQS), following turnover of the enzyme by addition of the substrate DAHP, gave a new crystal form (form J). Although the crystals have dimensions of only 50 x 20 x 5 micro m, they are well ordered, diffracting to 1.7 A. The space group is C222(1), with unit-cell parameters a = 90.0, b = 103.7, c = 177.4 A. Structure determination and refinement to R = 0.19 (R(free) = 0.25) shows the DHQS is in the 'open' form with the substrate site unoccupied but with some loop regions perturbed. Previous crystals of open-form DHQS only diffracted to 2.5 A resolution. The use of enzyme turnover may be applicable in other systems in attempts to improve crystal quality.

Tryptophan-plus-tyrosine double auxotrophic mutants resistant to fluorophenylalanine (PFP) and β-2-thienylalanine (TA) were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in mineral salts medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to 5-methyltryptophan (MT) and 3-nitrotyrosine (NT) from a double auxotrophic PFPr and TAr mutant. Under optimal cultural condition one mutant yielded 9.6g phenylalanine per L medium in flask culture. Enzymic activity of regulatory enzymes (deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase and prephenate dehydratase) were observed in the wild type, double auxotroph and double-auxotrophic multianalogue-resistant mutant.

Synthesis of 1-epi-dehydroquinate, 9, provided an authentic sample of this material and allowed its identification as a minor product in the noncatalyzed rearrangement of enolpyranose 4a to 3-dehydroquinate, 7 (3-DHQ). None of this isomer was detected in the product of the transformation of DAHP, 1, to 3-DHQ catalyzed by dehydroquinate synthase. This result indicates that the enolpyranose 4a is not released from the enzyme active site prior to rearrangement to 3-DHQ, a possibility suggested previously (Bartlett, P. A.; Satake, K. J. Am. Chem. Soc. 1988, 110, 1628-1630). Enolpyranose 4a was generated in the presence of DHQ synthase; however, the formation of 9 was not diminished, indicating that spontaneous rearrangement is faster than uptake by the enzyme under these condition. The question remains whether the enzyme takes an active role in catalyzing the rearrangement of 4a to 3-DHQ or simply provides a conformational template to prevent formation of theside product 9

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.

The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.

Epimeric carbaphosphonate [1R-(1α,3α,4β,5α)]-1,3,4-trihydroxy-5-(phosphonomethyl)cyclohexane-1-carboxylic acid and carbaphosphonate [1S-(1α,3β,4α,5β)]-1,3,4-trihydroxy-5-(phosphonomethyl)cyclohexane-1-carboxylic acid are potent inhibitors of purified 3-dehydroquinate (DHQ) synthase. Can either of these carbocyclic diastereomers inhibit DHQ synthase in intact plants? Obtaining sufficient amounts of both diastereomers for in vivo inhibition studies required the development of efficient synthetic routes to both molecules

The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.

Summary The present study aims to give a survey on common enzymes of the shikimate pathway in non-green tissues. Dehydroquinate hydrolyase/shikimate oxidoreductase, shikimate kinase and chorismate mutase from roots of spinach and a radish ( Brassica rapa ) hybrid were partially purified; the 3-dehydroquinate synthase was identified. Regarding some earlier findings of other authors and previewing current intermediate incorporation studies, it can be inferred that non-green tissues make use of the complete shikimate pathway to synthesize aromatic amino acids - unlike the pathway in photosynthetic tissues - independent from light.

Dehydroquinase, the third enzyme of the shikimate biosynthetic pathway, is inactivated by iodoacetate. Iodoacetate behaves as an affinity label for the Escherichia coli enzyme with a Ki of 30 mM and a limiting inactivation rate of 0.014 min-1 at pH 7.0 and 25 degrees C. Affinity labeling is mediated by the negative charge of the reagent since iodoacetamide does not inactivate the enzyme. 2.1-2.3 mol of carboxymethyl groups are incorporated per mol of protein monomer resulting in 90% inactivation of enzymic activity. The majority of the bound label (80%) is split equally between 2 methionine residues, Met-23 and Met-205, which were identified by sequencing radiolabelled peptide fragments isolated after proteolytic digestion. An equilibrium mixture of the substrate (dehydroquinate) and product (dehydroshikimate) substantially reduces the inactivation rate and specifically decreases the incorporation of label at both of these site, implicating them as being in or near the active site of the enzyme. Sequence alignments with other biosynthetic dehydroquinases show that of the 2 methionine residues only Met-205 is conserved. N-terminal alignments of all the available dehydroquinase sequences (both catabolic and biosynthetic classes) revealed that Met-23, although itself not conserved, resides within a cluster of conserved sequence which may constitute part of the dehydroquinate binding site. A consensus sequence was derived from these alignments and used to probe the protein sequence data banks. A related sequence was found in dehydroquinate synthase, the enzyme which precedes dehydroquinase in the shikimate pathway. These results suggest that we have identified part of the dehydroquinate binding site in both enzymes.

Dehydroquinate synthase (DHQS) has long been regarded as a catalytic marvel because of its ability to perform several consecutive chemical reactions in one active site1,2,3,4,5,6,7. There has been considerable debate as to whether DHQS is actively involved in all these steps1,2, or whether several steps occur spontaneously, making DHQS a spectator in its own mechanism3,4,5. DHQS performs the second step in the shikimate pathway, which is required for the synthesis of aromatic compounds in bacteria, microbial eukaryotes and plants8. This enzyme is a potential target for new antifungal and antibacterial drugs9,10 as the shikimate pathway is absent from mammals and DHQS is required for pathogen virulence11. Here we report the crystal structure of DHQS, which has several unexpected features, including a previously unobserved mode for NAD+-binding and an active-site organization that is surprisingly similar to that of alcohol dehydrogenase, in a new protein fold. The structure reveals interactions between the active site and a substrate-analogue inhibitor, which indicate how DHQS can perform multistep catalysis without the formation of unwanted by-products.

The aroB gene from Helicobacter pylori strain P1 was cloned and further characterized by sequence analysis and by functional complementation of the aroB mutation in Escherichia coli. The aroB gene encodes the enzyme 3-dehydroquinate synthase which catalyzes one of the early steps in the shikimate pathway. This pathway, which creates aromatic molecules from sugar precursors, is present in prokaryotes, fungi and plants but is absent from mammalian cells. The predicted amino acid sequence of the H. pylori aroB gene product showed significant homology (30-40% identity and 50-60% similarity) to 3-dehydroquinate synthases from various other prokaryotes and eukaryotes. The single gene on a plasmid was biologically active in E. coli. It suppressed the specific phenotype of aroB mutants by restoring the shikimate pathway-dependent synthesis of aromatic amino acids and the production of the siderophore enterobactin. Two other reading frames were found adjacent to the aroB gene. The first, designated as orf1, had no significant homology to proteins and genes present in databases, whereas the second was found to share a significant degree of homology with the tgt gene encoding tRNA-guanine transglycosylase from a variety of other bacteria (40-50% identity and 60-70% similarity). The function of the tgt gene was confirmed by heterologous complementation. The gene on a plasmid was shown to complement the queuosine biosynthesis defect in a genetically defined tgt- strain of E. coli. The presence of the aroB gene and the putative tgt homologue in unrelated H. pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers.

Phosphinomethyl and carboxymethyl monoacids along with succinyl, malonyl ether, malonyl, and hydroxymalonyl diacids were substituted for phosphorylmethyl, phosphonoethyl, and phosphonomethyl groups in carbocyclic inhibitors of DHQ synthase. All but one of the carbocyclic inhibitors were synthesized via intermediacy of a 2,3-butane bisacetal-protected 3-dehydroquinic acid. Carbaphosphinate (K(i) = 20 x 10(-)(6) M) was a modest competitive inhibitor of DHQ synthase, while carbaacetate was a linear mixed-type inhibitor (K(i) = 3 x 10(-)(6) M, K(i)' = 20 x 10(-)(6) M). Carbasuccinate (K(i) = 5 x 10(-)(6) M), carbamalonate ether (K(i) = 7 x 10(-)(6) M), carbamalonate (K(i) = 0.7 x 10(-)(6) M), and carbahydroxymalonate (K(i) = 0.3 x 10(-)(6) M) were all competitive inhibitors. Carbaacetate was the only inhibitor that was not oxidized by DHQ synthase. On the basis of these data, carbocyclic inhibitors with malonyl and hydroxymalonyl groups are apparently bound by DHQ synthase as tightly as carbocyclic inhibitors possessing phosphorylmethyl and phosphonoethyl moieties.

cDNA clones for all enzymes of the prechorismate pathway of higher plants have previously been cloned, with the exception of the second enzyme of the pathway, 3-dehydroquinate synthase. Here we describe the isolation of a cDNA encoding a 3-dehydroquinate synthase from tomato which was identified by complementing a 3-dehydroquinate synthase-deficient Escherichia coli strain with a tomato cDNA library. The deduced amino acid sequence contains a putative N-terminal plastid-specific transit peptide, and the sequence of the mature enzyme resembles those of the corresponding bacterial enzymes more than of the fungal enzymes. Sequence identity was even higher between the tomato and E. coli sequences than between the E. coli and other known bacterial sequences. The abundance of 3-dehydroquinate synthase transcripts differ in the organs of tomato plants analyzed. In cultured tomato cells, the abundance of 3-dehydroquinate synthase transcripts increased 9-fold within 4 to 5 h of elicitor treatment.

The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.

The aroL gene, encoding shikimate kinase of Brevibacterium lactofermentum, a coryneform glutamic acid-producing bacterium, was cloned. Recombinant plasmids containing the aroL gene caused elevated levels of shikimate kinase synthesis in B. lactofermentum. It was found that in addition to the aroL gene, the aroB and aroE genes, encoding dehydroquinate synthase and shikimate dehydrogenase, respectively, also existed on these recombinant plasmids, in complementation tests with various Escherichia coli and B. lactofermentum aromatic amino acid auxotrophs. The aroL, aroB and aroE genes of B. lactofermentum are located closely on the cloned DNA fragment, in that order. It was shown that at least these three aro genes form a cluster on the chromosome of B. lactofermentum.

Summary The following enzymes has been isolated of the yeast Hansenula henricii by means of ammonium sulfate fractionation, DEAE-Sephadex A-50 and Sepharose 6B column chromatography: 3-dehydroquinate synthase, 3-dehydroquinase-shikimate: NADP oxido-reductase complex, shikimate kinase, 5-enolpyruvyl shikimate-3-phosphate synthase, and quinate: NAD oxido-reductase. The approximate molecular weights of these enzymes have been estimated using gel filtration. Some properties of purified enzymes have been studied. Kinetic parameters and regulation of activity of enzymes were similar to those found in higher plants and bacteria. Aromatic amino acids do not regulate activity and level of the enzymes studied.

Dehydroquinate synthase has been purified 9000-fold from Escherichia coli K-12 (strain MM294). The synthase is encoded by the aroB gene, which is carried by plasmid pLC29-47 from the Carbon-Clarke library. Construction of an appropriate host bearing pLC29-47 results in a strain that produces 20 times more enzyme than strain MM294. Subcloning of the aroB gene behind a tac promoter results in E. coli transformants that produce 1000 times more enzyme than MM294: the synthase constitutes 5% of the soluble protein of the cell. A laborious isolation from 50 g of wild-type E. coli cells yields 80 micrograms of impure enzyme, whereas 50 g of cells containing the subcloned gene yields 150 mg of homogeneous enzyme in a two-column purification. Dehydroquinate synthase is a monomeric protein of Mr 40 000-44 000. The chromosomal enzyme from E. coli K-12, the cloned enzyme encoded by the plasmid pLC29-47, and the subcloned inducible enzyme encoded by pJB14 all comigrate on polyacrylamide gel electrophoresis under denaturing conditions.

3-Dehydroquinate synthase was purified to homogeneity from Escherichia coli. It was found to be a single polypeptide chain of Mr = approximately 57,000. Reaction mixtures of pure enzyme and the substrate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, were incubated for short times and treated with NaB3H4. The resulting 3-deoxyheptonic acid 7-phosphate was degraded with sodium periodate, and formic acid representing C-5 of the substrate was isolated. The presence of 3H in the formate corresponding to 15% of the enzyme was interpreted as indicating a 5-dehydro derivative of the substrate as an intermediate of the reaction. Quinic acid, resulting from reduction of 3-dehydroquinate with NaB3H4, was also isolated and degraded with periodate. The formate from C-4 of the quinate was unlabeled, indicating that 3,4-bisdehydroquinate is not an intermediate.

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.

The complete amino acid sequence of the Escherichia coli 3-dehydroquinate synthase has been determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli 3-dehydroquinate synthase is 362 amino acids long and has a calculated Mr of 38 880. Analysis of the aroB nucleotide sequence and its 5'- and 3'-flanking regions has identified the aroB promoter elements and a possible 3'-terminator site.

Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH 4 ) 2 SO 4 fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement.

Dehydroquinate synthase, an enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, was detected in cell-free extracts of etiolated Phaseolus mungo seedlings. The reaction product, 3-dehydroquinate, formed from [1-14C]DAHP was identified by paper-radiochromatography. The enzyme required NAD+ and Co2+ for activity.

The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.