Mutation analysis of the feedback inhibition site of phenylalanine‐sensitive 3‐deoxy‐<scp>D</scp>‐arabino‐heptulosonate 7‐phosphate synthase of <i>Escherichia coli</i>

Hu, Changyun; Pin, Jiang; Xu, Jianfeng; Wu, Yongqing; Wei-da, Huang

doi:10.1002/jobm.200310244

Cited by 34 publications

(31 citation statements)

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“…In particular the feedback inhibition of DAHP synthase (AroG, AroF) and CM/ PDT (PheA) has been studied by generation and characterization of fbr mutants (4,11,14,15,19,23). Those mutants were of particular interest due to the overproduction of L-phenylalanine by engineered bacteria (1).…”

Section: Discussionmentioning

confidence: 99%

Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) ofEscherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

Lütke‐Eversloh

Stephanopoulos

2005

Appl Environ Microbiol

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In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/ prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His 6 -tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA WT , but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA fbr mutants were similar to those of the TyrA WT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.Chorismate is a central intermediate of the shikimate pathway and branch point for five different metabolic pathways in microorganisms (10). The aromatic amino acids L-phenylalanine and L-tyrosine are formed from chorismate via prephenate, which undergoes either decarboxylation/dehydration or decarboxylation/dehydrogenation, followed by a transamination to form the respective amino acids (Fig. 1).Chorismate mutase/prephenate dehydrogenase (CM/PDH, TyrA) is a bifunctional enzyme occurring as a homodimer with a molecular weight of approximately 78,000 and is involved in tyrosine biosynthesis in Escherichia coli (13). The rearrangement of chorismate to prephenate is catalyzed by the N-terminal CM domain of TyrA, whereas the C-terminal PDH domain catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate. Analogously, the PDT domain of the bifunctional CM/PDT (PheA) yields phenylpyruvate, which is subsequently transaminated to phenylalanine.Aromatic amino acid biosynthesis is tightly regulated, in particular, at the chorismate branch point and at the first committed step, i.e., at 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate. The DAHP synthase occurs as three isoforms, each feedback regulated by either phenylalanine, tyrosine, or tryptophan (20). Furthermore, the TyrR protein, binding any one of the three aromatic amino acids, provides transcriptional control of eight different promoters related to aromatic amino acid biosynthesis (21).Due to the industrial importance of L-phenylalanine, for example, as a precursor for the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester), much more effort has been devoted to the investigation of the phenylalanine biosynthesis pathway than to the tyrosine branch (1). For the biotechnological production of phenylalanine, engineered E. coli strains were employed, exhibiting, among other characteristics, alleviated feedback inhibition by the end product (23, 24). As a consequence, different feedback-inhibition-...

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Section: Discussionmentioning

confidence: 99%

Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) ofEscherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

Lütke‐Eversloh

Stephanopoulos

2005

Appl Environ Microbiol

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“…Cloning and site-directed mutagenesis: The procedure for site-directed mutagenesis of aroG gene based on polymerase chain reaction (PCR) was described previously (HU et al 2003). The mutated genes were cloned into T vector and sequenced to confirm the desired mutation.…”

Section: Methodsmentioning

confidence: 99%

“…Determination of kinetic parameters: Enzymatic activity assay of AroG was described previously (HU et al 2003). For a typical reaction, 0.5 ml of 0.1 M phosphate buffer (pH 6.4) containing 25 nmol of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) was pre-incubated at 25. for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…Several amino acid substitutions in the N-terminal and interior region of polypeptide sequence of AroG, Asp146Asn, Pro150Leu (KIKUCHI et al 1997), Phe180Ser (GER et al 1994), Ile10Ala and Leu179Ala (HU et al 2003), Phe209Ser (JIANG et al 2000, and Val221Ala (HU et al 2003), have been reported to desensitize to feedback inhibition addressed by phenylalanine. Similarly, substitution of Pro18leu at the N-terminus of AroH results in a tryptophan-insensitive type of DAHPS, and an Asn8Lys-mutant and a truncation mutant AroF lacking the first seven amino acids lead to tyrosine-insensitive forms of DAHPS (JOSSEK et al 2001).…”

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confidence: 96%

“…Our previous work showed that single amino acid residue replacement (Ile10Ala) or deletion of the N-terminal 15 amino acids could lead to a dramatic loss of AroG enzymatic activity (HU et al 2003). Here we characterize the kinetic properties of these mutants and show that deletion of N-terminus prevents the enzyme from forming a dimeric structure, indicating that the N-terminus (1 -15 amino acids) of AroG plays a critical role in the formation of the essential tight dimmer, which associates into homotetramer in the cell.…”

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Requirement of the N‐terminus for dimer formation of phenylalanine‐sensitive 3‐deoxy‐D‐arabino‐heptulosonate synthase AroG of Escherichia coli

Shen

et al. 2004

J. Basic Microbiol.

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The first regulatory step in the synthesis of aromatic amino acids is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). In Escherichia coli, the allosteric DAHPS exists as three isozymes, AroG, AroF and AroH, each independently feedback-inhibited by corresponding end product amino acids, phenylalanine, tyrosine and typtophan. Structural biological evidences have suggested that the N-terminus of AroG is involved in the formation of a putative inhibitor-binding site and feedback inhibition signal transmission. Our previous work showed that a single amino acid residue replacement Ile10Ala or deletion of 15 N-terminal amino acids could lead to a dramatic loss of AroG enzymatic activity (Hu et al. 2003). Here we demonstrate that the deletion of N-terminus prevents the enzyme from forming a dimeric structure, indicating that the N-terminus of AroG plays a critical role in the formation of the essential tight dimeric structure.

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Shikimate Pathway and Aromatic Amino Acid Biosynthesis

Tzin

Galili

Aharoni

2012

Encyclopedia of Life Sciences

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The shikimate pathway consists of seven enzymatic reactions whose end product chorismate is the precursor for the synthesis of the aromatic amino acids Phe, Tyr and Trp. In fungi and plants, chorismate is a precursor for many specialised metabolites (i.e. secondary metabolites) that play an important role in the plant's interaction with its environment. The shikimate pathway and aromatic amino acid biosynthesis have been extensively studied in a variety of microorganisms, fungi and plants. Furthermore, the dual involvement of the shikimate and aromatic amino acid biosynthesis pathways in central and specialised metabolism still raises major questions regarding the genes and enzymes involved, and their control, their evolutionary origins and coordinated regulation with genes of associated pathways in response to altered environmental conditions and diverse developmental programs. Key Concepts: The shikimate pathway is the only known pathway for biosynthesis of chorismate and the aromatic amino acids Phe, Tyr and Trp. The shikimate pathway is a bridge between central metabolism and specialised metabolism. The shikimate pathway occurs in various groups of microorganisms, plants and parasites, whereas it does not occur in animals. The pathway enzymes are being targeted for antimicrobial drug and herbicide design. Shikimic acid is an essential metabolite that may balance the metabolic status of the pathway. Chorismate is a branch point metabolite for aromatic amino acids and phenolic compounds. This is an ancient eukaryotic pathway which has been subject to diverse evolutionary processes. Several enzymes from these pathways are allosterically regulated by their end products: Phe, Tyr or Trp. The shikimate pathway and aromatic amino acids, and the specialised metabolites derived from them, simultaneously respond to rhythmic changes.

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Mutation analysis of the feedback inhibition site of phenylalanine‐sensitive 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase of Escherichia coli

Cited by 34 publications

References 23 publications

Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) ofEscherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) ofEscherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

Requirement of the N‐terminus for dimer formation of phenylalanine‐sensitive 3‐deoxy‐D‐arabino‐heptulosonate synthase AroG of Escherichia coli

Shikimate Pathway and Aromatic Amino Acid Biosynthesis

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