Conservation of mechanism in three chorismate-utilizing enzymes.
Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromatic compounds in plants, bacteria, fungi, and some parasites. Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carry out the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, and enterobactin, respectively, and are expected to share a common mechanism. Poor binding to ADCS of two potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, like AS and IS, initiates reaction by addition of a nucleophile to C2. Molecular modeling based on the X-ray structures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed by ADCS to initiate the reaction, forming a covalent intermediate. The K274A and K274R mutants were shown to have 265- and 640-fold reduced k(cat) values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH(4)(+), ADCS and the K274A mutant have identical k(cat) values, suggesting the participation of NH(4)(+) as a rescue agent. Such participation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274A mutant but not ADCS, when either NH(4)(+) or PabA + glutamine is used as the nitrogen source. Additionally, the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate. A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 of chorismate in an S(N)2' ' process is proposed.
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Department of Chemistry, University of California-Davis, Davis, California 95616, USA.
Stigers Lavoie
Kimberly D
KD
Bartlett
Paul A
PA
Toney
Michael D
MD
D000878:Anthranilate Synthase / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D005021:Ethylamines / Q000737:chemistry / Q000378:metabolism; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005021:Ethylamines; D000637:Transaminases; C066599:aminodeoxychorismate synthase; D000878:Anthranilate Synthase; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid; C041564:ethylamine