p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase.

Ye, Qi Zhuang; Li, Jun; Walsh, Christopher T.

doi:10.1073/pnas.87.23.9391

Cited by 80 publications

(66 citation statements)

References 20 publications

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“…Nearly all work published on the enzymology of PABA biosynthesis has focused on the ammonia-dependent amination of chorismate by PabB, neglecting the contribution of PabA (16,29). Here we report kinetic data both on the glutamine amidotransferase activity of the PabB-PabA complex and on the ammonia-dependent activity, as catalyzed by the complex and by PabB alone.…”

Section: Discussionmentioning

confidence: 98%

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Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

1995

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The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB undergoes inactivation upon incubation at 37؇C, which is prevented by the presence of chorismate or PabA; glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the K m values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.

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Section: Discussionmentioning

confidence: 98%

“…After incubation at 37ЊC for 30 min, 50 l of the assay mix was applied to a Waters Nova-Pak C-18 4-m column (3.9 by 150 mm). The column was developed with 5% acetic acid, and the elution profile was monitored at 270 nm (9,29). Chorismate, PABA, and ADC eluted as distinct peaks.…”

Section: Methodsmentioning

confidence: 99%

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

1995

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“…ADC lyase, encoded by pabC, converts ADC to PABA and pyruvate (1, 6, 30, 31, * Corresponding author. 34). While in vitro data so far support a two-step reaction, it remains possible that the proteins exist in a ternary complex in vivo and that ADC synthase and ADC lyase remain active when separated.…”

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confidence: 99%

Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme

1992

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In Escherichia coli, p-aminobenzoate (PABA) is synthesized from chorismate and glutamine in two steps. Aminodeoxychorismate synthase components I and II, encoded by pabB and pabA, respectively, convert chorismate and glutamine to 4-amino-4-deoxychorismate (ADC) and glutamate, respectively. ADC lyase, encoded by pabC, converts ADC to PABA and pyruvate. We reported that pabC had been cloned and mapped to 25 min on the E. coli chromosome (J. M. Green and B. P. Nichols, J. Biol. Chem. 266:12971-12975, 1991). Here we report the nucleotide sequence of pabC, including a portion of a sequence of a downstream open reading frame that may be cotranscribed with pabC. A disruption of pabC was constructed and transferred to the chromosome, and the pabC mutant strain required PABA for growth. The deduced amino acid sequence of ADC lyase is similar to those of Bacillus subtilis PabC and a number of amino acid transaminases. Aminodeoxychorismate lyase purified from a strain harboring an overproducing plasmid was shown to contain pyridoxal phosphate as a cofactor. This finding explains the similarity to the transaminases, which also contain pyridoxal phosphate. Expression studies revealed the size of the pabC gene product to be approximately 30 kDa, in agreement with that predicted by the nucleotide sequence data and approximately half the native molecular mass, suggesting that the native enzyme is dimeric.

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“…The menaquinone and enterobactin pathways diverge following the conversion of chorismate to 2-hydroxy-4-deoxychorismate (isochorismate) by isochorismate synthase (28,30). For 4-aminobenzoate synthesis, chorismate is first aminated to form 4-amino-4-deoxychorismate, which then undergoes ,-elimination of the enol-pyruvyl moiety to form 4-aminobenzoate (1,8,21,29). The first step in ubiquinone biosynthesis is the conversion of chorismate to 4-hydroxybenzoate by chorismate lyase (5,15), in a reaction apparently similar to the second step of 4-aminobenzoate synthesis, catalyzed by 4-amino-4-deoxychorismate lyase.…”

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Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase

Nichols

Green

1992

J Bacteriol

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In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate. 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase. These two enzymes are encoded by ubiC and ubi4, respectively, and have been reported to map near one another at 92 min on the E. coli chromosome. We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubi4. The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubi4. ubiC was localized by subcloning, and overproducing plasmids were constructed. Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region. Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychorismate -. 4-aminobenzoate), the product of E. coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain. Of the several enzymes that convert chorismate to intermediates of E. coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized.Chorismate is the branch point precursor for the synthesis of many aromatic compounds in Escherichia coli. The seven major end products of chorismate anabolism are phenylalanine, tyrosine, tryptophan, ubiquinone, 4-aminobenzoate, menaquinone, and enterobactin. Chorismate itself undergoes five different conversions that result in those seven products. The tyrosine and phenylalanine pathways diverge following the isomerization of chorismate to prephenate, but two distinct, homologous chorismate mutase activities channel chorismate toward either phenylalanine or tyrosine (10). The menaquinone and enterobactin pathways diverge following the conversion of chorismate to 2-hydroxy-4-deoxychorismate (isochorismate) by isochorismate synthase (28,30). For 4-aminobenzoate synthesis, chorismate is first aminated to form 4-amino-4-deoxychorismate, which then undergoes ,-elimination of the enol-pyruvyl moiety to form 4-aminobenzoate (1,8,21,29). The first step in ubiquinone biosynthesis is the conversion of chorismate to 4-hydroxybenzoate by chorismate lyase (5, 15), in a reaction apparently similar to the second step of 4-aminobenzoate synthesis, catalyzed by 4-amino-4-deoxychorismate lyase. Finally, the first step in tryptophan synthesis is chorismate amination and p-elimination of the enol-pyruvyl group to form anthranilate, or 2-aminobenzoate (2).With the exception of the genes involved in ubiquinone biosynthesis, all of the E. coli genes encoding enzymes that use chorismate or 4-amino-4-deoxychorismate as substrates have been cloned and sequenced. These include pheA (chorismate mutase-prephenate dehydratase), tyrA (chorismate mutase-prephenate dehydrogenase) (10), entC (isochorismate synth...

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p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase.

Cited by 80 publications

References 20 publications

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme

Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase

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