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The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme.

We show that the antibacterial agent, (6S)-6-fluoroshikimate, is a substrate for the shikimate transport system of Escherichia coli because in exchange-diffusion experiments it displaced intracellular [14C]shikimate with the same kinetics as did unlabelled shikimate. Other shikimate analogues were also substrates: as judged by similar experiments or, in the case of (6R)-6-fluoroshikimate, by inference.

Quinic dehydrogenase, which catalyzes the conversion of quinic acid to 5-dehydroquinic acid, has been extracted from cells of an Aerobacter mutant and partially purified. The enzyme is DPN-specific. With initial extracts TPN also promotes the reactin, but its activity has been shown to be due to coupling with DPN by pyridine nucleotide transhydrogenase. Quinic dehydrogenase activity is conveniently measured by spectrophotometric determination of DPNH (using cyanide to inactivate DPNH oxidase). However, initial extracts also contain the necessary enzymes for further converting 5-dehydroquinic acid to 5-dehydroshikimic acid, and for reducing the latter compound (with TPNH) to shikimic acid. The coupling of this last reaction (via transhydrogenase) reoxidizes the DPNH formed by quinic dehydrogenase, and hence interferes with the spectrophotometric assay. This interference can be eliminated by removing TPN through pretreatment of the extract with charcoal, or by conventional methods of enzyme purification. Alternatively, enzyme activity can be determined by a bioassay method which is not affected by the coupled reaction. Certain properties of quinic dehydrogenase are described. No cofactor requirement (other than DPN) could be detected. In the series of coupled reactions, the overall equilibrium constant for shikimic/quinic is approximately i.o. The limited distribution of quinic dehydrogenase, compared with the broader distribution of known enzymes of aromatic biosynthesis, supports the conclusion that quinic acid is probably not an intermediate in this biosynthetic sequence.

Agaricus bisporus contains novel aromatic compounds. By incubation of the mushroom with [G-14 C]shikimic acid, the radioactivity was incorporated into tyrosine, phenylalanine and several unidentified metabolites. The most radioactive metabolite in the stipe and the cap was identified as N-(gamma-L-glutamyl)-4-hydroxyaniline. The radioactivity was proved to be localized in the 4-hydroxyaniline moiety of this compound.

14C-labelled shikimic acid and double labelled shikimic acid tritiated stereospicifically at C-6 are incorporated into 3-(3-carboxyphenyl) alanine, 3-(3-carboxy-4-hydroxyphenyl) alanine, phenylalanine, and tyrosine in Reseda lutea L., Reseda odorata L., Iris x Hollandica cv. Prof. Blauw, and Iris x hollandica cv. Wedgwood. The experiments with 14C-labelled shikimic acid confirm that the aromatic carboxyl groups and rings in 3-(3-carboxyphenyl) alanine and 3-(3-carbocy-4-hydroxyphenyl) alanine derive from the carbocyl group and ring in shikimic acid whereas the experiments with double labelled shikimic acid demonstrate that the pro-6S-hydrogen atom is retained and the pro-6R-hydrogen atom lost in the biosynthesis of 3-(3-carboxyphenyl) alanine, phenylalanine, and tyrosine in the plants used. 3H was located in the ortho-position in the aromatic rings of phenylalanine and tyrosine but in a position para to the alanine side chain of 3- (3-carboxyphenly) alanine. No 3H was found in 3- (3-carboxy-4-hydroxyphenyl) alanine. This supports a derivation of the last two compounds from chorismic acid via isochorismic acid, isoprephenic acid, and 3'-carboxyphenylpyruvic acid and 3'-carbocy-4'-hydroxyphenylphruvic acid. The 3H/14C ratio in 3-(3-carboxyphenyl) alanine was found higher than in the precursor used. This isotope effect must operate by competition between the pathways from isoprephenic acid to 3'-carbocyphenylpyruvic acid and to 3'-carbocy-4'- hydroxyphenylpyruvic acic. The proposed biosynthetic pathways for the two carboxy-substituted amino acids are in agreement with their distribution patterns in the plant kingdom and suggest that they may derive from minor changes of enzymes involved in the general pathways of aromatic biosynthesis.

Two associated enzymes, 3-dehydroquinate hydro-lyase (EC 4.2.1.10) and shikimate:NADP+ oxidoreductase (EC 1.1.1.25), have been purified from Phaseolus mungo seedlings. These enzymes were purified 6900- and 9700-fold, respectively, but they were not separable. Moreover, two activity bands of the shikimate:NADP+ oxidoreductase were detected after polyacrylamide gel electrophoresis and the two peaks also have 3-dehydroquinate hydro-lyase activity. The two forms of the associated enzymes showed only small differences in molecular weight, Km value, pH optimum and the responses to some inhibitors.

1. 1. A new growth phase of Escherichia coli B in the presence of p -fluorophenylalanine is described. This is a slow exponential growth phase which follows the linear growth induced by the analog, and which continues for several generations until supplanted by a resistant mutant. A graphic analysis of the complete growth cycle is given. 2. 2. The p -fluorophenylalanine is incorporated exponentially during the slow exponential growth, without alteration, as a partial substitute for phenylalanine. 3. 3. Extensive filamentation of the organisms occurs during linear growth. Cell division is resumed in the slow exponential phase. 4. 4. The phenomena of inhibition of mass increase and inhibition of cell division are shown to be separable. Cell-division inhibition appears dependent on tyrosine deprivation or interference with tyrosine metabolism. It is postulated that dl -p- fluorophenylalanine interferes with aromatic biosynthesis by inhibiting enzymes early in the pathway and by partially repressing the synthesis of these enzymes. 5. 5. A new interpretation is made of the nature of the resistant mutant.

1. 1. Aerobacter aerogenes NCW (wild-type strain) was grown on quinic acid in either iron-sufficient or iron-deficient media. Catechol, 2,3- and 3,4-dihydroxybenzoic acids, p-hydrobenzoic acid and anthranilic acid accumulated in iron-deficient medium whereas only the latter two acids where found in iron-sufficient medium. 2. 2. A. aerogenes N5-36 (a mutant blocked after the synthesis of anthranilic acid) when grown on quinic acid accumulated catechol, 2,3- and 3,4-dihydroxybenzoic acids, p-hydrobenzoic acid, anthranilic acid and an N-acylanthranilic acid. This latter compoun which was not the N-formyl derivative had similar characteristics to N-acetylanthranillic acid. 3. 3. A washed suspension of cells of the mutant grown either on glucose or quinic acid also produced this N-acylanthranilic acid together with anthranilic acid when incubated with shikimic acid. 4. 4. The possibilities of this N-acyl derivative being either a precursor or a metabolite of anthranilic acid are discussed.

1. 1. Fangman and Neidhardt 1,2 used mutant Escherichia coli strains unable to activate p-fluorophenylalanine (PFPA) to show that amino acid activation is required for ribonucleic acid synthesis. Their findings were confirmed. 2. 2. It was shown directly that the mutant cells do not charge phenylalanine-specific transfer ribonucleic acid with PFPA. 3. 3. A possible criticism of the interpretation is that PFPA might create a shortage of tyrosine and tyrptohan by false feedback inhibition of 3-deoxy-d- arabino -heptulosonic acid 7-phosphate synthetase, and inhibit ribonucleic acid synthesis thereby, rather than by failure of PFPA activation. PFPA inhibition in the mutant was indeed reversed by shikimate, and therefore presumably affects the 3-deoxy- d - arabino -heptulosonic acid 7-phosphate synthetase and decrease the supply of shikimate. However, in the mutant cultures, the limitation of ribonucleic acid synthesis occuring in presence of PFPA was not overcome by supplementation with tyrosine and tryptophan at concentrations adequate for permeation in presence of PFPA. 4. 4. It is concluded that amino acid activation is required for ribonucleic acid synthesis.

When Neurospora grows in media supplemented with tryptophan, 70–90% of the supplied tryptophan is converted in a series of reactions to anthranilic acid or derivatives of anthranilic acid. In certain strains (those blocked between shikimic acid and anthranilic acid), these reactions, coupled with the biosynthetic reactions, constitute a cycle involving the interconversion of anthranilic acid and tryptophan. Studies on the physiological control of these reactions revealed a marked effect of temperature upon the efficiency of utilization of tryptophan for the synthesis of protein. Mutants blocked after anthranilic acid gave considerably less growth when cultured at 23% than when cultured at 33°. Mutants blocked anthranilic acid gave nearly equal amounts of growth at either temperature. Experiments with isotopes ruled out the possibility that altered rates of flow of tryptophan through certain reactions of the cycle were causing the observed effects. Subsequent investigation revealed that a small but significant quantity of indolepyruvic acid was accumulated in filtrates of tryptophan-less mutants grown at the higher temperature and that this compound supported growth of strains requiring tryptophan. Studies with cell-free extracts of Neurospora indicate that the interconversion of tryptophan and indolepyruvic acid is mediated by a constitutive transaminase. These and earlier results permit a clearer interpretation of the complex interplay between genes, enzymes and substrates in the metabolism of tryptophan by Neurospora.

A study has been made of the control by end-product inhibition of the tryptophan biosynthetic pathway in cultured cells of the higher plant, Nicotiana tabacum , var. Wisconsin 38. Crude extracts of callus tissue obtained from N. tabacum catalyzed the conversion of chorismic acid to anthranilic acid, the first step unique to l -tryptophan version of chorismic acid to anthranilic acid, the first step unique to l -tryptophan biosynthesis. The catalytic activity was destroyed by heat and no reaction occured with the ommission of either glutamine of Mg 2+ . In cell extracts this catalytic activity was inhibited by l -tryptophan. The inhibition was competitive with chorismate. In experiments with intact cells the presence of high l -tryptophan pools reduced the flow of radioactive carbon from the precursor, shikimic acid, into biosynthetic L -tryptophan. The data obtained were consistent with the operation of end-product inhibition in vivo

A number of auxotrophs ofAerobacter aerogenes requiring aromatic amino acids for growth have been shown to excrete o-dihydric phenols when incubated with a mixture of glucose, phosphate, magnesium and ammonium ions. Auxotrophs requiring histidine, methionine, cysteine or leucine do not excrete these substances under similar conditions. An auxotroph requiring tyrosine, phenylalanine and tryptophan for growth but unable to grow on shikimic acid accumulates a mixture of 3,4-dihydroxy-benzoic acid and catechol. A tryptophan auxotroph able to utilise anthranilic acid for growth accumulates 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid and catechol. A tyrosine auxotroph able to utilise 4-hydroxyphenylpyruvic acid has been shown to accumulate 2,3-dihydroxybenzoic acid and catechol. Catechol may arise consequent on 3,4-dihydroxybenzoic acid accumulation since suspensions of wild typeA. aerogenes form catechol from 3,4-dihydroxybenzoic acid. Such suspensions also metabolise 3,4-dihydroxybenzoic acid and catechol to compounds not possessing the o-dihydric phenol structure when aerated in the absence of glucose. l -Tryptophan inhibits the accumulation of o-dihydric phenols by three different tryptophan auxotrophs ofA. aerogenes but has no effect on the accumulation of these compounds by a tyrosine auxotroph or an auxotroph requiring tyrosine, phenylalanine and tryptophan for growth. The possible relationship of these o-dihydric phenols to the biosynthesis of aromatic compounds is discussed.

An improved method is described for the purification of the arom multi-enzyme complex from Neurospora crassa. The product of purification is an aggregate of approximately 230 000 mol. wt having five enzyme activities which function in the common pathway in aromatic amoni acid biosynthesis. Conditions promoting dissociation of the enzyme complex has been determined. Alkaline pH values, storage in low ionic-strength buffers, and high temperature all promote dissociation. The aggregate fragments resulting from dissociation are quite labile and have not been thoroughly characterized. However, in all but one of the fragments observed 5-dehydroshikimate reductase (Shikimate: NADP+ oxidoreductase, EC 1.1.1.25) and 5-dehydroquinate dehydratase (5-dehydroquinate hydro-lyase, EC 4.2.1.10) activities are associated. These two activities have been found alone or in association with one of the three additional activities. A presumptive dimer of 3-enolpyruvylshikimic acid 5-phosphate synthelase has also been observed. To explain the variety of fragments observed, the minimum number of polypeptides present is postulated to be 4. Thus, the possibility that the 115 000 mol. wt subunits of the complex are single multifunctional polypeptide has been excluded.

2,3-Dihydroxybenzoate has been shown to be a growth factor in shaken cultures of certain multiple aromatic auxotrophs of Escherichia coli and Aerobacter aerogenes . The requirement for 2,3-dihydroxybenzoate is influenced by the presence of iron and citrate in the medium. The effects of the latter compounds differ with an auxotrophic strain derived from E. coli W and auxotrophic strains derived from E. coli K12. In the case of the former strain, certain metal ions or anaerobic growth will eliminate the 2,3-duhydroxybenzoate requirement; in the latter strains citrate replaces 2,3-dihydroxybenzoate. 2,3-Dihydroxybenzoate is formed via the shikimic acid pathway from chrismate in E. coli and A. aerogenes . The enzyme system forming 2,3-dihydroxybenzoate from choristmate in A. aerogenes has been studied and shown to require NAD and Mg 2+ for activity. At least two steps appear to be involved in the overall reaction. The enzyme system converting chorismate into 2,3-dihydroxybenzoate is strongly repressed by low concentrations of iron or cobalt and is also affected by the presence of salicylate or citrate in the growth medium.

Synthesis of salicylic acid has been studied in washed cell suspensions and cell-free extracts of Mycobacterium smegmatis grown under iron-deficient conditions. The pathway of biosynthesis is from shikimate via chorismate and finally isochorismate. The trivial name salicylate synthetase is proposed for the enzyme catalysing the final step from isochorismate. Apparently co-factors are not required and 2,3-dihydro-2,3-dihydroxybenzoate is not an intermediate in this reaction. Biosynthesis of salicylate from isochorismate has also been demonstrated in cell-free extracts from Mycobacterium tuberculosis BCG and Mycobacterium fortuitum (both of which contain salicylate in their mycobactins) but not in extracts from Mycobacterium phlei (which contains only 6-methylsalicylate). Enzymes synthesizing salicylate from chorismate or isochorismate were repressed by about 95% when M. smegmatis was grown in iron-sufficient (2 μg iron per ml) medium. Phenolic acids, including salicylate, did not repress enzyme activity. Iron (as FeCl 3 or ferrimycobactin), desferrimycobactin or salicylate did not inhibit the activities of the enzymes. Decline of salicylate synthetase activity during iron-deficient growth of M. smegmatis was closely correlated with the rate of mycobactin formation in the cell and with salicylate disappearance from the media.

The transformation of quinic acid into shikimic acid by means of the methylene derivatives of these acids has been described. Thus the configuration of the carbon atoms 3,4 and 5 of quinic acid has been shown to be the same as in shikimic acid, which had previously been configurationally related to D -glucose. 3-cyano-lobelanine has been synthesized from dihydroshikimic acid nitrile, benzoyl acetic acid, and monomethyl amine under conditions sufficiently mild so that they might exist in plant or animal organisms.

1. 1. In Escherichia coli , β-2-thienylalanine drastically inhibited protein synthesis within 5 min. Shikimic acid reversed the inhibition non-competitively. The non-competitive nature of the inhibition was confirmed both by measuring the internal concentration of thienylalanine and by estimating it from the rate of thienylalanine incorporation into protein. 2. 2. The 3-deoxy- d - arabino -heptulusonic acid 7-phosphate (DAHP) synthetase normally inhibited by phenylalanine is the principal one present in exponentially growing cells in minimal medium. It was strongly inhibited by thienylalanine. There was no inhibition of the tyrosine-controlled DAHP synthetase. 3. 3. A mutant selected for resistance to thienylalanine had a normal amount of DAHP synthetase. About half of the enzyme was the tyrosine-repressible species, and the other half was insensitive to inhibition by phenylalanine or tyrosine, or repression by tyrosine or tryptophan. 4. 4. The K m for phenylalanine with phenylalanyl-RNA synthetase was 6·10 −6 M. The K i for thienylalanine inhibition of phenylalanine activation was approx. 2·10 −4 M. Although thienylalanine was incorporated into protein, transfer of thienylalanine to transfer RNA by the phenylalanyl-RNA synthetase could not be demonstrated in vitro . 5. 5. The data indicate that false feedback inhibition of the phenylalanine-sensitive DAHP synthetase was responsible for the immediate inhibition of protein synthesis by thienylalanine.