• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.238-245
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• 1988

Studies were made on the regulation of chorismate mutase and prephenate dehydratase of a species of Intrasporangium, a phenylalanine producing Actinomycete isolated from soil. Two distinctly regulated species of chorismate mutase, designated CM I and CM IIwere resolved by DEAE Cellulose and DEAE Sephadex A 50 chromatography. The activity of CM II was inhibited by L-tyrosine, whereas that of CM I appeared to be unregulated. Single species of prephenate dehydyatase was also separated in the same purification steps. The activity of the enzyme was strongly feedback inhibited by L-phenylalanine, but by L-tyrosine or L-methionine it was rather slightly stimulated. Synthesis of chorismate mutase was not influenced by the presence of phenylalanine, tyrosine or tryptophan, whereas prephenate dehydratase was found to be subject to strong feedback repression by L-phenylalanine. The rate of repression was 94% at the concentration of 1mM L-phenylalanine but the repression was completely offset by the presence of 5mM tyrosine. The critical regulatory site of the phenylalanine terminal biopathway was, therefore, proved to be the second reaction which was catalyzed by the L-phenylalanine inhibitable and repressible prephenate dehydratase.

• BMB Reports
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• v.34 no.4
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• pp.299-304
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• 2001

The 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase is the first enzyme of aromatic amino acid-, folic acid-, and phenazine-1-carboxylic acid biosynthetic pathways. DAHP synthase of Bacillus sp. B-6 that produces phenazine-1-carboxylic acid was feedback inhibited by two intermediary metabolites of aromatic amino acid biosynthetic pathways, prephenate and chorismate, but not by other metabolites, such as anthranilic acid, shikimic acid, p-aminobenzoic acid, and 3-hydroxyanthranilic acid. DAHP synthase of Bacillus sp. B-6 was not inhibited by end products, such as aromatic amino acids, folic acid, and phenazine-1-carboxylic acid. The inhibition of DAHP synthase by prephenate and chorismate was non-competitive with respect to erythrose 4-phosphate and phosphoenolpyruvate. Prephenate and chorismate inhibited 50% of the DAHP synthase activity at concentrations of $2{\times}10^{-5}\;M$ and $1.2{\times}10^{-4}\;M$, respectively The synthesis of DAHP synthase of Bacillus sp. B-6 was not repressed by exogenous aromatic amino acids, folic acid, and phenazine 1-carboxylic acid, single or in combinations.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.174-179
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• 1990

In order to overproduce L-phenylalanine, various kind of regulatory mutants were isolated from parental Escherichia coli K-12. MWEC 83 Producing 7.4g/l of L-phenylalanine has been derived as a tyrosine and tryptophan double auxotrophic mutant. To produce L-phenylalanine without adding L-tyrosine and L-tryptophan, revertant strain MWEC 101 was isolated from MWEC 83. Further various analogues and valine resistant mutants were isolated from MWEC 101. MWEC 101-5 was the most excellent strain that produced 17.9g/l of L-phenylalanine after having been cultivated for 54 hours in 15% glucose medium. It was disclosed that activities of rate-limiting enzymes including chorismate mutase and prephenate dehydratase in MWEC 101-5 were desensitized to 2mM L-phenylalanine in the enzyme reaction mixture and that activities level of 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase and prephenate dehydratase were increased more than 20 times over those of the parental strain.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.278-285
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• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.135-153
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• 2015

The aromatic amino acids, which are composed of $\small{L}$-phenylalanine, $\small{L}$-tyrosine and $\small{L}$-tryptophan, are general components of protein synthesis as well as precursors for a wide range of secondary metabolites. These aromatic amino acids-derived compounds play important roles as ingredients of diverse phenolics including pigments and cell walls, and hormones like auxin and salicylic acid in plants. Moreover, they also serve as the natural products of alkaloids and glucosinolates, which have a high potential to promote human health and nutrition. The biosynthetic pathways of aromatic amino acids share a chorismate, the common intermediate, which is originated from shikimate pathway. Then, tryptophan is synthesized via anthranilate and the other phenylalanine and tyrosine are synthesized via prephenate, as intermediates. This review reports recent studies about all the enzymatic steps involved in aromatic amino acid biosynthetic pathways and their gene regulation on transcriptional/post-transcriptional levels. Furthermore, results of metabolic engineering are introduced as efforts to improve the production of the aromatic amino acids-derived secondary metabolites in plants.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.169-173
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• 1990

For the overproduction of L-phenylalanine using Escherichia coli, the authors constructed various recombinant plasmids including pMW 10, pMW 11 and pMW 12. The $aroF{FR}$ and $pheA^{FR}$ genes for the production of L-phenylalanine were isolated from Escherichia coli MWEC 101-5 strains. The productivity and atability of Escherichia coli regulatory mutants containing recombinant plasmids were investigated to evaluate the efficiency of the $aroF^{FR}$ and $pheA^{FR}$ genes. The MWEC 101-5/pMW 11 strain produced 24.3g/l of L-phenylalanine while its stability was 73.8 percent. The specific activity of prephenate dehydratase in the MWEC 101-5/pMW 11 strain increased by 26-fold compared with that of Escherichia coli K-12.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.119-127
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• 1985

A series of Escherichia coli mutants were exmined for ability to convert glucose and ammonium salts into phenylalanine. This enabled the biochemical changes having major. effects on phenylaianine yield, and interactions between mutations, to be identified. Changes to the common pathway of aromatic biosynthesis having a major effects include desensitization of the first enzyme (3-deoxy-D-arabinoheptulosonate synthase) to end-product inhibition, and removal of repression of enzyme synthesis. It is suggested that the 3-deoxy-D-arabino-heptulosonate synthase Phe isoenzyme has a more important effect on yield. Similarly, removal of repression and end-product inhibition on the phenylalanine terminal pathway increased yield, and changes to both common and branch pathways were synergistic. Blockage of the typrosine and tryptophan pathways had minor effects on phenylalanine yield, and a mutation affecting aramatic amino acid transport (aroP) decreased yield. With multiple-mutation strains hish specific rates of product formation (ie 0.1-0.17g phenylalanine/g cells/h) were obtained.