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PubMed1987

Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants.

5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2024

Revealing novel protein interaction partners of glyphosate in Escherichia coli.

Despite all debates about its safe use, glyphosate remains the most widely applied active ingredient in herbicide products, with renewed approval in the European Union until 2033. Non-target organisms are commonly exposed to glyphosate as a matter of its mode of application, with its broader environmental and biological impacts remaining under investigation. Glyphosate displays structural similarity to phosphoenolpyruvate (PEP), thereby competitively inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), crucial for the synthesis of aromatic amino acids in plants, fungi, bacteria, and archaea. Most microbes, including the gut bacterium Escherichia coli (E. coli), possess a glyphosate-sensitive class I EPSPS, making them vulnerable to glyphosate's effects. Yet, little is known about glyphosate's interactions with other bacterial proteins or its broader modes of action at the proteome level. Here, we employed a quantitative proteomics and thermal proteome profiling (TPP) approach to identify novel protein binding partners of glyphosate in the E. coli proteome. Glyphosate exposure significantly altered amino acid synthesizing pathways. The abundance of shikimate pathway proteins was increased, suggesting a compensatory mechanism. Extracellular riboflavin concentrations were elevated upon glyphosate exposure, while intracellular levels remained stable. Beyond the target enzyme EPSPS, thermal proteome profiling indicated an effect of glyphosate on the thermal stability of certain proteins, including AroH and ProA, indicating interactions. Similar to the competitive binding between PEP and glyphosate at EPSPS, one reason for the interaction of AroH and ProA with the herbicide could be a high structural similarity between their substrates and glyphosate. Overall, glyphosate induced metabolic disturbances in E. coli, extending beyond its primary target, thereby providing new insights into glyphosate's broader impact on microbial systems.

Glyphosate↗
PubMed1995

The Metabolism of Quinate in Pea Roots (Purification and Partial Characterization of a Quinate Hydrolyase).

A quinate (QA) hydrolyase was isolated from pea (Pisum sativum L.) roots. The enzyme converts QA into shikimate by elimination of water. The enzymatic reaction is independent of cofactors and divalent cations. The QA hydrolyase was purified about 1,600-fold to apparent electrophoretic homogeneity in three steps, including bovine serum albumin-affinity chromatography. The enzyme forms oligomers and/or complexes with bovine serum albumin and ovalbumin. The monomer molecular weight of the enzyme is about 15,000. The hydrolyase shows regular Michaelis-Menten kinetics with a Km, of 2.0 mM for QA. Compartmentation studies reveal that the QA hydrolyase is localized in plastids. The QA hydrolyase may function in channeling imported QA into the shikimate pathway to support aromatic amino acid biosynthesis in plastids.

Journal Article↗
PubMed2000

Salicylates of intact Salix myrsinifolia plantlets do not undergo rapid metabolic turnover.

Salicylates, the main phenolic glucosides of northern willow (Salix spp.), play an important role in plant-herbivore interactions. Salicylates are labile metabolites that are thought to undergo metabolic turnover. Salicylates are synthesized from phenylalanine (Phe) via the shikimate pathway. 2-Aminoindan-2-phosphonic acid (AIP), a strong inhibitor of Phe ammonia-lyase (EC 4.3.1.5), was used to block the biosynthesis of salicylates. The aim of this study was to investigate long-term turnover of salicylates in intact micropropagated plantlets of Salix myrsinifolia Salisb. The biosynthesis of salicylates was inhibited efficiently but not completely by 30 microM 2-aminoindan-2-phosphonic acid. Inhibitor treatment, aside from leading to a high accumulation of Phe, also led to an increase in tyrosine and tryptophan, indicating that 2-aminoindan-2-phosphonic acid may also inhibit enzymes other than Phe ammonia-lyase. Salicylates were shown to be unexpectedly stable metabolites that did not undergo marked metabolic turnover in intact plants; in leaves no significant turnover occurred, and in the stems the five salicylates studied were turned over slowly, with half-lives of 11 to 25 d. The total amount of salicylate in mature shoots decreased only 0.6% per day.

Amino Acids, Cyclic↗
PubMed2001

Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, the starter unit of rifamycin biosynthesis in amycolatopsis Mediterranei S699.

To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.

Actinomycetales↗
PubMed1975

Relationship between the tonB locus and iron transport in Escherichia coli.

When a strain (arcB-) of Escherichia coli, unable to synthesize the iron transport compound enterochelin, was transduced to tonB-, it became resistant to phage phi80 and simultaneously lost the growth response to enterochelin and the ability to transport its iron complex. However, enterochelin precursors (shikimate and 2,3-dihydroxybenzoate) still supported growth, via the synthesis of enterochelin. Dihydroxybenzoate was a better growth factor at a low concentration than it was at higher levels. The evidence suggests that tonB- strains lack an outer membrane component necessary both for the uptake of ferric-enterochelin and for the adsorption of phage phi80. Thus, although ferric-enterochelin cannot penetrate the cell surface from outside, the complex that is formed within the envelope is transported normally into the cell. The aroB-, tonB- mutant also lacked growth reponses to citrate and various hydroxamate siderochromes, which supported growth in the tonB+ parent strain via inducible transport systems for their ferric complexes. The aroB-, tonB- mutant was unable to transport iron in the presence of citrate, but the low-affinity uptake of uncomplexed iron and the transport of amino acids and phosphate were unimpaired. The tonB locus, thus, affects all the known active transport systems for iron, possibly indicating that they share some common outer membrane component.

Amino Acids↗
PubMed1993

Solid-state NMR determination of intra- and intermolecular 31P-13C distances for shikimate 3-phosphate and [1-13C]glyphosate bound to enolpyruvylshikimate-3-phosphate synthase.

Rotational-echo, double-resonance (REDOR) 31P NMR was used to obtain internuclear distances for shikimate 3-phosphate (S3P) and N-(phosphonomethyl)-[1-13C]glycine (glyphosate) bound to 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a 46-kDa enzyme essential for the synthesis of aromatic amino acids in plants and microorganisms. An intermolecular 31P-13C distance of 7.2 A was measured between the phosphate of S3P and the labeled carbon of glyphosate. This means that S3P and glyphosate are in proximity in the binding site of the enzyme. An intramolecular 31P-13C distance of 5.6 A was measured between the phosphonate 31P and the labeled carbon of glyphosate. This distance can be achieved only if glyphosate is completely extended when bound to EPSP synthase.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

5-Enolpyruvyl shikimate 3-phosphate synthase from Escherichia coli. Identification of Lys-22 as a potential active site residue.

5-Enolpyruvyl shikimate 3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enolpyruvyl shikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). In order to determine the role of lysine residues in the mechanism of action of this enzyme as well as in its inhibition by glyphosate, chemical modification studies with pyridoxal 5'-phosphate were undertaken. Incubation of the enzyme with the reagent in the absence of light resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order and saturation kinetics with Kinact of 45 microM and a maximum rate constant of 1.1 min-1. The inactivation rate increased with increase in pH, with a titratable pK of 7.6. Activity of the inactive enzyme was restored by addition of amino thiol compounds. Reaction of enzyme with pyridoxal 5'-phosphate was prevented in the presence of substrates or substrate plus glyphosate, an inhibitor of the enzyme. Upon 90% inactivation, approximately 1 mol of pyridoxal 5'-phosphate was incorporated per mol of enzyme. The azomethine linkage between pyridoxal 5'-phosphate and the enzyme was reduced by NaB3H4. Tryptic digestion followed by reverse phase chromatographic separation resulted in the isolation of a peptide which contained the pyridoxal 5'-phosphate moiety as well as 3H label. By amino acid sequencing of this peptide, the modified residue was identified as Lys-22. The amino acid sequence around Lys-22 is conserved in bacterial, fungal, as well as plant enzymes suggesting that this region may constitute a part of the enzyme's active site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2004

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1993

A novel operon organization involving the genes for chorismate synthase (aromatic biosynthesis pathway) and ribosomal GTPase center proteins (L11, L1, L10, L12: rplKAJL) in cyanobacterium Synechocystis PCC 6803.

Many of the ribosomal protein (RP) genes in both bacterial and chloroplast genomes occur, for reasons not yet understood, in operons that include nonribosomal genes. Here we report such an operon organization in a cyanobacterium (Synechocystis PCC6803) involving the genes for four RPs that are important in the GTPase function of the ribosome and the aroC gene encoding chorismate synthase, a key enzyme in the shikimate pathway for biosynthesis of aromatic amino acids and cell wall components. The Synechocystis aroC encodes a 362-amino-acid residue protein which is 52, 60, and 68% identical to two eubacterial (both 52%), yeast, and a higher plant (Corydalis) chorismate synthase, respectively. The gene was overexpressed in Escherichia coli, and the gene product was shown to cross-react with antibodies to Corydalis chorismate synthase; it also complemented an aroC-lacking E. coli strain. The Synechocystis rpl1 and rpl11 genes encode polypeptides of 237 and 141 amino acid residues, respectively, also with high sequence identities to the corresponding RP sequences from other eubacteria and higher plant chloroplasts. The gene order is shown to be: rpl11-86bp spacer-rpl1-460bp spacer-rpl10-87-bp spacer-rpl12-206bp spacer-aroC. Southern and Northern blot analyses of Synechocystis DNA and RNA, respectively, revealed a single cluster of these genes per genome which is transcribed from a common promoter to an unusually long, approximately 9500-nucleotide transcript. Several constructs of the cyanobacterial aroC and rpl12 genes were made and expressed in E. coli to examine the mechanisms for their very differential expression from a polycistronic mRNA (e.g. four copies L12/ribosome; chorismate synthase, a non-abundant protein). These results present the first biochemical/molecular genetic evidence of shikimate pathway in the cyanobacterial group.

Amino Acid Sequence↗
PubMed2000

Simultaneous analysis of shikimate-derived secondary metabolites in Lithospermum erythrorhizon cell suspension cultures by high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) analysis system based on a water-acetonitrile gradient program was established for simultaneous quantification of shikimate-derived secondary metabolites in cultured cells of Lithospermum erythrorhizon. The cells cultured in pigment production medium (M-9) are capable of producing five highly hydrophilic compounds such as p-hydroxybenzoic acid-O-glucoside and lithospermic acid B, as well as eleven lipophilic compounds including echinofuran B and acetylshikonin. In addition to the wide polarities of those compounds, many of them are unstable under light, dryness, oxygen and heating. Thus, a new extraction procedure for all these compounds was also established by use of ultrasonication under ice-water chilling with MeOH as the solvent. This procedure was applied to the quantitative analyses of these compounds in cell cultures and hairy root cultures of Lithospermum, and in the intact plants as well.

Anthraquinones↗
PubMed2004

Overexpression of a cyanobacterial phosphoenolpyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis changes amino acid metabolism.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C4-like pathway is proposed.

Amino Acids↗
PubMed1976

Growth and synthesis of rubratoxin by Penicillium rubrum in a chemically defined medium fortified with organic acids and intermediates of the tricarboxylic acid cycle.

A sterile glucose-mineral salts broth was fortified with equimolar concentrations (10--3 M) of various organic acids and intermediates in the tricarboxylic acid cycle. Appropriate media were neutralized with 2 N NaOH, inoculated with spore suspensions or mycelial pellets of Penicillium rubrum and incubated quiescently for 14 days or with shaking for 5 days. Rubratoxins were recovered from culture filtrates by ether extraction and resolved by thin-layer chromatography. Toxin formation in quiescent cultures was enhanced by malonate but was not markedly affected by ethyl malonate, shikimate, and acetate or by isocitrate or oxaloacetate added in the presence of malonate. Citrate, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, and malonate when present in the medium alone or in conjunction with malonate caused a 15 to 50% reduction in rubratoxin formation. Acetyl-CoA (10--5 M/flask) caused an 80% increase in toxin yield. Rubratoxin formation in shake cultures was not affected by succinate and malonate. All other combinations of intermediates and malonate caused a 10 to 50% reduction in toxin formation. At 10--3 M, citrate enhanced rubratoxin B formation and stimulated rubratoxin A production by as much as 100%. Above 10--3 M, citrate inhibited toxin production. Incorporation of [2-14C]acetate into rubratoxin was enhanced by malonate, fumarate, and malonate. A combination of pyruvate and malonate produced a 40% increase in [2-14C]acetate incorporation into rubratoxin. The highest reduction of labeled acetate incorporation (36%) was caused by succinate or alpha-ketoglutarate combined with malonate.

Acetates↗
PubMed2003

Identification of the catalytic residues of AroA (Enolpyruvylshikimate 3-phosphate synthase) using partitioning analysis.

AroA (EPSP synthase) catalyzes carboxyvinyl transfer through addition of shikimate 3-phosphate (S3P) to phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (THI), followed by phosphate elimination to give enolpyruvylshikimate 3-phosphate (EPSP). A novel approach, partitioning analysis, was used to elucidate the roles of catalytic residues in each step of the reaction. Partitioning analysis involved trapping and purifying [1-(14)C]THI, degrading it with AroA, and quantitating the products. Wild-type AroA gave a partitioning factor, f(PEP) = 0.25 +/- 0.02 at pH 7.5, where f(PEP) = [[1-(14)C]PEP]/([[1-(14)C]PEP] + [[1-(14)C]EPSP]). Eighteen mutations were made to 14 amino acids to discover which residues preferentially catalyzed either the addition or the elimination step. Mutating a residue catalyzing one step (e.g., addition) should change f(PEP) to favor the opposite step (e.g., elimination). No mutants caused large changes in f(PEP), with experimental values from 0.07 to 0.41. This implied that there are no side chains that catalyze only addition or elimination, which further implied that the same residues are general acid/base catalysts in both forward and reverse THI breakdown. Only Lys22 (protonating S3P hydroxyl or phosphate) and Glu341 (deprotonating C3 of PEP) are correctly situated in the active site. In the overall reaction, Lys22 would act as a general base during addition, while Glu341 would act as a general acid. Almost half of the mutations (eight of 18) caused a >1000-fold decrease in specific activity, demonstrating that a large number of residues are important for transition state stabilization, "ensemble catalysis", in contrast to some enzymes where a single amino acid can be responsible for up to 10(8)-fold catalytic enhancement.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2002

How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli.

The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP synthase from Escherichia coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP synthase and corroborates the assumption that glyphosate and PEP target the same binding site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMedDate not supplied

[The possibilities of GC/MS and HPLC in the analysis of sugars and acids in natural matrices].

A GC/MS procedure is described for the simultaneous quantitation of the minor and major constituents of several natural matrices, including fruits, vegetables, mushrooms, drug/industrial plants and honeys, as their trimethylsilyl derivatives, from one solution, by one injection. Selected minor components have been determined on the basis of their characteristic fragment ions, in the presence of extremely high excess of saccharides of various degree of polymerization. Selective fragmentation of these minor compounds in the ion trap detector provided possibilities for distinguishing them. In the case of honeys the method permitted the simultaneous quantitation of o-phosphoric, malic, shikimic, citric/isocitric, quinic, margaric, oleic and stearic acids, 5-hydroxymethylfurfurol and proline with the extremely high sugar contents of honeys (fructose, glucose, galacturonic acid, inositol, sucrose, trehalose, turanose, maltose, gentiobiose, isomaltose, raffinose, erlose, melezitose, maltotriose, panose, isomaltotriose) and allowed the fast evaluation of sugar and acid constituents of fifteen honeys from various floral and geological origin. Results revealed that (i) the minor components varied in the concentration range of 0.0001 to 0.43%, and, (ii) together with the saccharides of honeys made up the total of identified and determined constituents from 87.8% to 98.5%. Quantitative evaluation of the minor constituents was performed on the basis of their selective fragment ion values with an average reproducibility of 6.7% (RSD).

Biological Factors↗
PubMed1998

Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer--sequence of the gene and deduced structural properties of the enzyme.

The gene coding for the periplasmic quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa ATCC 17933 was cloned and sequenced. The deduced amino acid sequence contained a signal peptide of 34 residues and the major protein of 589 amino acids showed high similarities to pyrroloquinoline-quinone-dependent periplasmic and membrane-bound dehydrogenases acting on alcohols, glucose and quinate or shikimate. It was demonstrated by alignment with the amino acid sequence of the large subunit of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens, whose X-ray structure is known, that the amino acid residues involved in the binding of pyrroloquinoline quinone and Ca2+ at the active site are conserved in the quinoprotein ethanol dehydrogenase of P. aeruginosa. Also, the glycine/tryptophan docking motifs involved in stabilizing the superbarrel structure of the quinoprotein methanol dehydrogenase of M. extorquens were conserved. The known sequences of pyrroloquinoline-quinone-dependent dehydrogenases were used to derive new, more specific sequence motifs for detecting members of this family of enzymes. Despite the sequence similarity between the large a subunit of quinoprotein methanol dehydrogenase from M. extorquens and the quinoprotein ethanol dehydrogenase from P. aeruginosa, the two enzyme systems were quite different. In the presence of the prosthetic group, pyrroloquinoline quinone expression of the Pseudomonas gene encoding the 60-kDa subunit of quinoprotein ethanol dehydrogenase in Escherichia coli resulted in formation of active enzyme. The formation of active quinoprotein methanol dehydrogenase, however, is known to require, in addition to the large alpha subunit, the expression of a small beta subunit, and helper proteins [Lidstrom, M. E. (1995) Genetics of bacterial quinoproteins, Methods Enzymol. 258, 217-227].

Alcohol Oxidoreductases↗
PubMed2001

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme-ligand complexes in solution, we have isolated the 26-kDa N-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate-3-phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N-terminal domain upon S3P binding.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗