Conservation of Mechanism in Three Chorismate-Utilizing Enzymes

Ze He, Kimberly D. Stigers Lavoie, Paul A. Bartlett, Michael D. Toney

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

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 kcat values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH4 +, ADCS and the K274A mutant have identical kcat values, suggesting the participation of NH4 + 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 NH4 + 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 N2" process is proposed.

Original languageEnglish (US)
Pages (from-to)2378-2385
Number of pages8
JournalJournal of the American Chemical Society
Volume126
Issue number8
DOIs
StatePublished - Mar 3 2004

Fingerprint

Anthranilate Synthase
Nucleophiles
Conservation
Enzymes
Biosynthesis
Glutamine
Nitrogen
para-Aminobenzoates
Enterobactin
Molecular modeling
Aromatic compounds
Fungi
Tryptophan
Catalytic Domain
Bacteria
Parasites
X-Rays
Derivatives
X rays
isochorismate synthase

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

He, Z., Stigers Lavoie, K. D., Bartlett, P. A., & Toney, M. D. (2004). Conservation of Mechanism in Three Chorismate-Utilizing Enzymes. Journal of the American Chemical Society, 126(8), 2378-2385. https://doi.org/10.1021/ja0389927

Conservation of Mechanism in Three Chorismate-Utilizing Enzymes. / He, Ze; Stigers Lavoie, Kimberly D.; Bartlett, Paul A.; Toney, Michael D.

In: Journal of the American Chemical Society, Vol. 126, No. 8, 03.03.2004, p. 2378-2385.

Research output: Contribution to journalArticle

He, Z, Stigers Lavoie, KD, Bartlett, PA & Toney, MD 2004, 'Conservation of Mechanism in Three Chorismate-Utilizing Enzymes', Journal of the American Chemical Society, vol. 126, no. 8, pp. 2378-2385. https://doi.org/10.1021/ja0389927
He, Ze ; Stigers Lavoie, Kimberly D. ; Bartlett, Paul A. ; Toney, Michael D. / Conservation of Mechanism in Three Chorismate-Utilizing Enzymes. In: Journal of the American Chemical Society. 2004 ; Vol. 126, No. 8. pp. 2378-2385.
@article{15e04d9ffc65432289ad67fa6633b6f6,
title = "Conservation of Mechanism in Three Chorismate-Utilizing Enzymes",
abstract = "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 kcat values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH4 +, ADCS and the K274A mutant have identical kcat values, suggesting the participation of NH4 + 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 NH4 + 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 N2{"} process is proposed.",
author = "Ze He and {Stigers Lavoie}, {Kimberly D.} and Bartlett, {Paul A.} and Toney, {Michael D.}",
year = "2004",
month = "3",
day = "3",
doi = "10.1021/ja0389927",
language = "English (US)",
volume = "126",
pages = "2378--2385",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Conservation of Mechanism in Three Chorismate-Utilizing Enzymes

AU - He, Ze

AU - Stigers Lavoie, Kimberly D.

AU - Bartlett, Paul A.

AU - Toney, Michael D.

PY - 2004/3/3

Y1 - 2004/3/3

N2 - 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 kcat values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH4 +, ADCS and the K274A mutant have identical kcat values, suggesting the participation of NH4 + 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 NH4 + 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 N2" process is proposed.

AB - 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 kcat values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH4 +, ADCS and the K274A mutant have identical kcat values, suggesting the participation of NH4 + 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 NH4 + 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 N2" process is proposed.

UR - http://www.scopus.com/inward/record.url?scp=1542306930&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1542306930&partnerID=8YFLogxK

U2 - 10.1021/ja0389927

DO - 10.1021/ja0389927

M3 - Article

C2 - 14982443

AN - SCOPUS:1542306930

VL - 126

SP - 2378

EP - 2385

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 8

ER -