Abstract
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 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 Km 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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- Bauerle R., Hess J., French S. Anthranilate synthase-anthranilate phosphoribosyltransferase complex and subunits of Salmonella typhimurium. Methods Enzymol. 1987;142:366–386. doi: 10.1016/s0076-6879(87)42049-1. [DOI] [PubMed] [Google Scholar]
- Buvinger W. E., Stone L. C., Heath H. E. Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans. J Bacteriol. 1981 Jul;147(1):62–68. doi: 10.1128/jb.147.1.62-68.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Caligiuri M. G., Bauerle R. Identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. Evidence for an amino-terminal regulatory site. J Biol Chem. 1991 May 5;266(13):8328–8335. [PubMed] [Google Scholar]
- Goncharoff P., Nichols B. P. Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase. J Bacteriol. 1984 Jul;159(1):57–62. doi: 10.1128/jb.159.1.57-62.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green J. M., Merkel W. K., Nichols B. P. Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme. J Bacteriol. 1992 Aug;174(16):5317–5323. doi: 10.1128/jb.174.16.5317-5323.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green J. M., Nichols B. P. p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC. J Biol Chem. 1991 Jul 15;266(20):12971–12975. [PubMed] [Google Scholar]
- Huang M., Gibson F. Biosynthesis of 4-aminobenzoate in Escherichia coli. J Bacteriol. 1970 Jun;102(3):767–773. doi: 10.1128/jb.102.3.767-773.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kane J. F., Holmes W. M., Jensen R. A. Metabolic interlock. The dual function of a folate pathway gene as an extra-operonic gene of tryptophan biosynthesis. J Biol Chem. 1972 Mar 10;247(5):1587–1596. [PubMed] [Google Scholar]
- Kaplan J. B., Nichols B. P. Nucleotide sequence of Escherichia coli pabA and its evolutionary relationship to trp(G)D. J Mol Biol. 1983 Aug 15;168(3):451–468. doi: 10.1016/s0022-2836(83)80295-2. [DOI] [PubMed] [Google Scholar]
- McLeish M. J., Caine J. M. Chemical modification of PABA synthase. Biochem Int. 1991 Aug;24(6):1033–1042. [PubMed] [Google Scholar]
- McLeish M. J., Wookey P. J., Mortimer K. G. The cloning and over-expression of PABA synthase in E. coli. Biochem Int. 1988 Apr;16(4):727–735. [PubMed] [Google Scholar]
- Nichols B. P., Seibold A. M., Doktor S. Z. para-aminobenzoate synthesis from chorismate occurs in two steps. J Biol Chem. 1989 May 25;264(15):8597–8601. [PubMed] [Google Scholar]
- Roux B., Walsh C. T. p-Aminobenzoate synthesis in Escherichia coli: mutational analysis of three conserved amino acid residues of the amidotransferase PabA. Biochemistry. 1993 Apr 13;32(14):3763–3768. doi: 10.1021/bi00065a031. [DOI] [PubMed] [Google Scholar]
- Roux B., Walsh C. T. p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA. Biochemistry. 1992 Aug 4;31(30):6904–6910. doi: 10.1021/bi00145a006. [DOI] [PubMed] [Google Scholar]
- Sawula R. V., Crawford I. P. Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation. J Bacteriol. 1972 Nov;112(2):797–805. doi: 10.1128/jb.112.2.797-805.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ye Q. Z., Liu J., Walsh C. T. p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9391–9395. doi: 10.1073/pnas.87.23.9391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zalkin H. Anthranilate synthetase. Adv Enzymol Relat Areas Mol Biol. 1973;38:1–39. doi: 10.1002/9780470122839.ch1. [DOI] [PubMed] [Google Scholar]
- Zalkin H. The amidotransferases. Adv Enzymol Relat Areas Mol Biol. 1993;66:203–309. doi: 10.1002/9780470123126.ch5. [DOI] [PubMed] [Google Scholar]