Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants.
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.
Yoshimura
Tokiko
T
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
Sagami
Ikuko
I
Sasakura
Yukie
Y
Shimizu
Toru
T
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D006418:Heme / Q000737:chemistry*; D007477:Ions; D007501:Iron / Q000737:chemistry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry
D007477:Ions; D006418:Heme; D000242:Cyclic AMP; D007501:Iron; D010727:Phosphoric Diester Hydrolases