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Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase.

The Escherichia coli gene murZ, encoding the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase, has been cloned and sequenced. Identified by screening an E. coli genomic library for clones that conferred phosphomycin resistance, murZ encoded a 419-amino-acid polypeptide and was mapped to 69.3 min on the E. coli chromosome. MurZ protein was purified to near homogeneity and found to have the expected UDP-N-acetylglucosamine enolpyruvyl transferase activity. Sequence analysis of the predicted product revealed 44% identity to OrfR from Bacillus subtilis (K. Trach, J.W. Chapman, P. Piggot, D. LeCoq, and J.A. Hoch, J. Bacteriol. 170:4194-4208, 1988), suggesting that orfR may also encode a UDP-N-acetylglucosamine enolpyruvyl transferase enzyme. MurZ is also homologous to the aromatic amino acid biosynthetic enzyme enolpyruvyl shikimate phosphate synthase, the other enzyme known to catalyze an enolpyruvyl transfer.

Alkyl and Aryl Transferases↗

Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori.

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.

Amino Acid Sequence↗

The shikimate pathway and its branches in apicomplexan parasites.

The shikimate pathway is essential for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Seven enzymes of the shikimate pathway catalyze sequential conversion of erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate is then used as a substrate for other pathways that culminate in production of folates, ubiquinone, napthoquinones, and the aromatic amino acids tryptophan, phenylalanine, and tyrosine. The shikimate pathway is absent from animals and present in the apicomplexan parasites Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Inhibition of the pathway by glyphosate is effective in controlling growth of these parasites. These findings emphasize the potential benefits of developing additional effective inhibitors of the shikimate pathway. Such inhibitors may function as broad-spectrum antimicrobial agents that are effective against bacterial and fungal pathogens and apicomplexan parasites.

Amino Acid Sequence↗

Biosynthesis of phenazine pigments in mutant and wild-type cultures of Pseudomonas aeruginosa.

Pigmentation mutants of Pseudomonas aeruginosa, selected by observed visual differences in coloration from the wild-type strain, were examined for altered patterns of phenazine synthesis. Three classes of mutants that were incapable of pyocyanine production were identified. Pigmentation patterns that were found to characterize the various mutant classes implicated precursor-product relationships, and a biochemical scheme covering the terminal reactions of pyocyanine biosynthesis is proposed. Among compounds tested as inhibitors of pigmentation, two effectively inhibited pyocyanine production production while allowing cell growth. p-Aminobenzoate inhibited total pigmentation; i.e., no other phenazine accumulated. m-Aminobenzoate inhibited a presumptive methylation step in pyocyanine biosynthesis, abolishing the formation of pyocyanine and aeruginosin pigments but increasing the yields of phenazine 1-carboxylic acid and oxychlororaphin. D-[2,3,4,5(n)-14C]shikimate was most efficiently incorporated into phenazines in the middle to late exponential phase of growth. Label was incorporated predominantly into pyocyanine in the absence of inhibitors and into phenazine 1-carboxylic acid when the organism was grown in the presence of m-aminobenzoate.

4-Aminobenzoic Acid↗

Cloning and analysis of the shiA gene, which encodes the shikimate transport system of escherichia coli K-12.

In Escherichia coli K-12, the shiA gene is involved in the uptake of shikimate. This gene has been cloned and its nucleotide sequence determined. The gene is predicted to encode a protein of 438 amino acids and lies adjacent to the amn gene. The hydropathy profile and the amino acid sequence indicate that the ShiA protein is a polytopic membrane protein that shows a homology with members of the major facilitator superfamily of transport proteins. Recombining an inactive form of the cloned gene into the chromosome creates mutants unable to transport shikimate. Introducing a wild-type gene on a multicopy plasmid into a shiA mutant restores the ability to transport shikimate. When this multicopy shiA plasmid is introduced into an aroE strain, this strain is now able to grow with shikimate as the aromatic supplement, consistent with the notion that dehydroshikimate (DHS) accumulated in an aroE strain prevents uptake of shikimate by competition. Expression of the shiA gene does not appear to be regulated by the TyrR protein, a repressor/activator that controls the expression of other genes involved with the biosynthesis or transport of the aromatic amino acids.

Amino Acid Sequence↗

Biosynthetic studies on the origin of the cyclohexanecarboxylic acid moiety of ansatrienin A and omega-cyclohexyl fatty acids.

Feeding experiments with [2,6,10,10-2H4]chorismate in Streptomyces collinus (ansatrienin A) and Alicyclobacillus acidocaldarius (omega-cyclohexyl fatty acids), and inhibitor experiments with glyphosate in the latter organism, have shown that the biosynthesis of cyclohexanecarboxylic acid branches off from the shikimate pathway at a point prior to enolpyruvylshikimate 3-phosphate, either at shikimate or shikimate 3-phosphate.

Bacillaceae↗

Studies on the biosynthesis of the alpha-glucosidase inhibitor acarbose: valienamine, a m-C7N unit not derived from the shikimate pathway.

Feeding experiments with Actinoplanes sp. SN223/29 showed that 3-amino-5-hydroxy-[7-13C]benzoic acid is not incorporated into acarbose (I). The valienamine moiety of I is thus not derived in the same way, from the shikimate pathway, as the m-C7N units in the ansamycin, mitomycin and ansamitocin antibiotics. Feeding experiments with [U-13C3]-glycerol followed by analysis of I by multiple quantum NMR spectroscopy support this conclusion and point to formation of the valienamine moiety by cyclization of a heptulose phosphate which arises from a triose phosphate via successive transfer of two 2-carbon fragments by transketolase, as proposed by Pape and co-workers.

Acarbose↗

The Mycobacterium tuberculosis shikimate pathway genes: evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases.

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.

Amino Acid Sequence↗

New developments in oxidative fermentation.

Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto- D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto- D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40 degrees C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38-40 degrees C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37 degrees C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateuriiCHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.

Acetic Acid↗

The biosynthesis of shikimate metabolites.

This review covers the literature published during 2000 on the biosynthesis of compounds derived wholly or partly from intermediates on the shikimate pathway. Recent developments in the enzymology and genetics of the shikimate pathway arc also described. Enzymes involved in the biogenetic pathway to the aromatic amino acids are covered initially followed by sections dedicated to metabolites derived in some part from intermediates on the pathway. These include pyrrolnitrin. violacein. indole-3-acetic acid, coumarins, lignans, lignin, tannins, melanin, flavanoids, ubiquinone, TOPA quinone, PQQ, and tropanes.

Amino Acids↗

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway.

[5-3H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB3H4 and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem. 15, 428-435). The [5-3H]shikimate has been converted enzymatically to [5-3H]chorismate and [5-3H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatographic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.

Chorismic Acid↗

The Site of the Inhibition of the Shikimate Pathway by Glyphosate: II. INTERFERENCE OF GLYPHOSATE WITH CHORISMATE FORMATION IN VIVO AND IN VITRO.

In the presence of the nonselective herbicide glyphosate (N-[phosphonomethyl]glycine), buckwheat (Fagopyrum esculentum Moench) hypocotyls and cultured cells of Galium mollugo L. accumulate an organic acid, which was identified as shikimate by mass-spectroscopy of its methyl ester. After growth in 0.5 millimolar glyphosate for 10 days, G. mollugo cells contained shikimate in amounts of up to 10% of their dry weight. Synthesis of chorismate-derived anthraquinones in G. mollugo was blocked by glyphosate. Chorismate and o-succinylbenzoate (an anthraquinone precursor) alleviated the inhibition. The conclusion drawn from these experiments, that glyphosate inhibits a step in the biosynthetic sequence from shikimate to chorismate, was substantiated by the finding that glyphosate is a powerful inhibitor of the conversion of shikimate to chorismate in cell-free extracts from Aerobacter aerogenes 62-1.

Journal Article↗

Shikimate and folate pathways in the protozoan parasite, Perkinsus olseni.

We have exploited the experimental accessibility of the protozoan parasite Perkinsus olseni and its similarities to apicomplexan parasites to investigate the influence of specific drugs on its proliferation. For this purpose, shikimate and folate pathways present an attractive target for parasitic therapy given their major differences with mammalian pathways. Glyphosate, a potent inhibitor of the shikimate pathway enzyme EPSP synthase inhibited the in vitro proliferation of P. olseni in a dose-dependent manner and this effect was reversed by addition of chorismate, indicating the presence of a shikimate pathway. However, this effect was not antagonised by p-aminobenzoate or folic acid. Furthermore, antagonism was observed, via pyrimethamine to glyphosate inhibitory effect, suggesting that the shikimate pathway is not essential for the biosynthesis of folate precursors and is therefore crucial for another pathway downstream from chorismate. In addition, sulfadiazine, a well known inhibitor of dihydropteorate synthase, an enzyme of the folate biosynthetic pathway,had no inhibitory effect on P. olseni proliferation. In view of these results, the parasite does not appear to require the folate biosynthesis pathway for its survival and is most likely able to use exogenous folate. Even though pyrimethamine was found to inhibit P. atlanticus growth, this inhibitory effect could not be reversed by co-addition of folic acid. Therefore, we propose that the effect of pyrimethamine observed in this study results from the inhibition of a target other than dihydrofolate reductase. Similarly, proguanil target is likely to be separate from DHFR since only its metabolite cycloguanil has been shown to have inhibitory properties on DHFR.

Animals↗

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase.

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.

Adenosine Diphosphate↗

Identification of 4-amino-4-deoxychorismate synthase as the molecular target for the antimicrobial action of (6s)-6-fluoroshikimate.

(6S)-6-Fluoroshikimate has antimicrobial activity. The molecular basis of this effect had not been identified, but there was speculation that (6S)-6-fluoroshikimate is first converted in vivo into 2-fluorochorismate, which then could inhibit 4-amino-4-deoxychorismate synthase (ADCS). 2-Fluorochorismate was prepared from E-fluorophosphoenolpyruvate and erythose-4-phosphate by the sequential reactions of DAHP synthase, dehydroquinate synthase, dehydroquinase, shikimate dehydrogenase, EPSP synthase, and chorismate synthase. Inhibition studies on ADCS showed that it was inhibited rapidly and irreversibly by 2-fluorochorismate. Electrospray mass spectrometry of the inactivated enzyme showed an additional mass of 198 +/- 10 Da. A novel peptide of 1087.6 Da was identified in the HPLC trace for the tryptic digest of 2-fluorochorismate-inactivated ADCS. Sequencing of this peptide by MS/MS showed that the peptide corresponded to residues 272-279 with a modification of 206.1 Da on Lys-274. This observation is particularly exciting in the context of a recent proposal for the catalytic mechanism of ADCS.

Anti-Infective Agents↗