Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase.
Anthranilate synthase [chorismate pyruvatelyase (amino-accepting), E.C.4.1.3.27] catalyzes the formation of anthranilate (o-aminobenzoate) and pyruvic acid from chorismate and glutamine. A mutant form of the enzyme from Salmonella typhimurium accumulates a compound that we had isolated and identified as trans-6-amino-5-[(1-carboxyethenyl)-oxy]-1,3- cyclohexadiene-1-carboxylic acid, commonly called aminodeoxyisochorismate (ADIC). Here we report that ADIC is formed by a reversible, Mg(2+)-dependent ADIC synthase activity of anthranilate synthase that can be functionally uncoupled from a Mg(2+)-dependent ADIC lyase activity of the enzyme by single amino acid substitutions in the TrpE subunit of the anthranilate synthase complex of S. typhimurium. Both of the component activities of the enzyme are sensitive to feedback inhibition by L-tryptophan. Purified ADIC is quantitatively converted to anthranilate and pyruvic acid by the ADIC lyase activity of wild-type anthranilate synthase. ADIC also serves as a substrate for the formation of chorismate by the enzyme in the absence of glutamine and (NH4)2SO4. The rate of ADIC formation by the mutant enzyme and the steady-state parameters for ADIC utilization by the wild-type enzyme are consistent with a role for ADIC as an enzyme-bound intermediate that does not accumulate during the course of the anthranilate synthase reaction. The altered catalytic specificity of mutant anthranilate synthase enzymes suggests a potential role for ADIC in secondary metabolism.
Morollo
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AA
Department of Biology, University of Virginia, Charlottesville 22903.
Bauerle
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R
D000878:Anthranilate Synthase / Q000096:biosynthesis / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000878:Anthranilate Synthase