PMID:14551206
| Citation |
Yoshimura, T, Sagami, I, Sasakura, Y and Shimizu, T (2003) Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants. J. Biol. Chem. 278:53105-11 |
|---|---|
| Abstract |
The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme. |
| Links |
PubMed Online version:10.1074/jbc.M304408200 |
| Keywords |
Binding Sites; Catalysis; Catalytic Domain; Chromatography, Gel; Cyclic AMP/metabolism; Dimerization; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Gene Deletion; Heme/chemistry; Ions; Iron/chemistry; Models, Biological; Mutagenesis, Site-Directed; Mutation; Oxidation-Reduction; Phosphoric Diester Hydrolases/chemistry; Phosphoric Diester Hydrolases/metabolism; Plasmids/metabolism; Protein Binding; Protein Structure, Tertiary; Spectrophotometry |
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