Structure-function relationships of EcDOS, a heme-regulated phosphodiesterase from Escherichia coli.

Recent studies have revealed a new class of heme enzymes, the heme-based sensors, which are able to turn on or off cellular signal transduction pathways in response to environmental changes. One of these enzymes is the heme-regulated phosphodiesterase from Escherichia coli (EcDOS). This protein is composed of an N-terminal heme-containing PAS domain and a C-terminal functional domain.

DOS(Ec), a heme-regulated phosphodiesterase, plays an important role in the regulation of the cyclic AMP level in Escherichia coli.

Heme-regulated phosphodiesterase from Escherichia coli (DOS(Ec)) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOS(Ec) was originally cloned from E.

Investigation of the relationship between protein-protein interaction and catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) by protein microarray.

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS.

Protein microarray system for detecting protein-protein interactions using an anti-His-tag antibody and fluorescence scanning: effects of the heme redox state on protein-protein interactions of heme-regulated phosphodiesterase from Escherichia coli.

A highly sensitive microarray system for detecting protein-protein interactions has been developed. This method was successfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS). To immobilize (His)6-Tag fused Ec DOS, anti-(His)6-Tag monoclonal antibody (anti-(His)6-Tag mAb) was initially immobilized on the solid surface, and (His)6-Tag fused Ec DOS was fixed by antigen-antibody interactions.

Critical roles of Asp40 at the haem proximal side of haem-regulated phosphodiesterase from Escherichia coli in redox potential, auto-oxidation and catalytic control.

In haem-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS), haem is bound to the PAS domain, and the redox state of the haem iron regulates catalysis by the PDE domain. We generated mutants of Asp40, which forms a hydrogen bond with His77 (a proximal haem axial ligand) via two water molecules, and a salt bridge with Arg85 at the protein surface. The redox potential of haem was markedly increased from 67 mV vs.