Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production
| Autor: | Michael D. Toney, Justin E. Culbertson |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Aminodeoxychorismate synthase
Stereochemistry Biophysics Gene Expression Biochemistry Catalysis Analytical Chemistry chemistry.chemical_compound Pyocyanin Bacterial Proteins Biosynthesis Escherichia coli Chorismic acid Molecular Biology Transaminases Glutamine amidotransferase ATP synthase biology Recombinant Proteins chemistry Pseudomonas aeruginosa Pyocyanine biology.protein Isochorismate synthase Phenazines Anthranilate synthase |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1834:240-246 |
| ISSN: | 1570-9639 |
| DOI: | 10.1016/j.bbapap.2012.10.010 |
| Popis: | 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 Mg 2 + dependent and catalyzes a similar S N 2" 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 K chorismate = 20 ± 4 μM, K Mg 2 + = 294 ± 22 μM, K L-gln = 11 ± 1 mM, and k cat = 2.2 ± 0.2 s − 1 for a random kinetic mechanism. PhzE can use NH 4 + as an alternative nucleophile, while Co 2 + and Mn 2 + are alternative divalent metals. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect