Expression and characterization of PhzE from P. aeruginosa PAO1

Aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1- carboxylate production

Justin E. Culbertson, Michael D. Toney

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2 + dependent and catalyzes a similar S N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give Kchorismate = 20 ± 4 μM, KMg 2 + = 294 ± 22 μM, KL-gln = 11 ± 1 mM, and kcat = 2.2 ± 0.2 s- 1 for a random kinetic mechanism. PhzE can use NH4 + as an alternative nucleophile, while Co2 + and Mn2 + are alternative divalent metals.

Original languageEnglish (US)
Pages (from-to)240-246
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1834
Issue number1
DOIs
StatePublished - Jan 2013

Fingerprint

Pyocyanine
Anthranilate Synthase
Enzymes
Ammonia
Kinetics
Phenazines
Nucleophiles
Salicylates
Cloning
Biosynthesis
Virulence Factors
Sequence Homology
Pseudomonas
Glutamine
Pyruvic Acid
Pseudomonas aeruginosa
Escherichia coli
Purification
Organism Cloning
Substitution reactions

Keywords

  • Aminodeoxyisochorismate
  • Chorismic acid
  • PhzE
  • Pseudomonas aeruginosa
  • Pyocyanin

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

Cite this

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title = "Expression and characterization of PhzE from P. aeruginosa PAO1: Aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1- carboxylate production",
abstract = "PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2 + dependent and catalyzes a similar S N2{"} nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give Kchorismate = 20 ± 4 μM, KMg 2 + = 294 ± 22 μM, KL-gln = 11 ± 1 mM, and kcat = 2.2 ± 0.2 s- 1 for a random kinetic mechanism. PhzE can use NH4 + as an alternative nucleophile, while Co2 + and Mn2 + are alternative divalent metals.",
keywords = "Aminodeoxyisochorismate, Chorismic acid, PhzE, Pseudomonas aeruginosa, Pyocyanin",
author = "Culbertson, {Justin E.} and Toney, {Michael D.}",
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T2 - Aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1- carboxylate production

AU - Culbertson, Justin E.

AU - Toney, Michael D.

PY - 2013/1

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N2 - PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2 + dependent and catalyzes a similar S N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give Kchorismate = 20 ± 4 μM, KMg 2 + = 294 ± 22 μM, KL-gln = 11 ± 1 mM, and kcat = 2.2 ± 0.2 s- 1 for a random kinetic mechanism. PhzE can use NH4 + as an alternative nucleophile, while Co2 + and Mn2 + are alternative divalent metals.

AB - PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg2 + dependent and catalyzes a similar S N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give Kchorismate = 20 ± 4 μM, KMg 2 + = 294 ± 22 μM, KL-gln = 11 ± 1 mM, and kcat = 2.2 ± 0.2 s- 1 for a random kinetic mechanism. PhzE can use NH4 + as an alternative nucleophile, while Co2 + and Mn2 + are alternative divalent metals.

KW - Aminodeoxyisochorismate

KW - Chorismic acid

KW - PhzE

KW - Pseudomonas aeruginosa

KW - Pyocyanin

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