Biochemistry Z 0250 Structure-Function Relationships of EcDOS, a Heme-Regulated Phosphodiesterase from Escherichia coli -[65 refs.]. -(SASAKURA*, Y.; YOSHIMURA-SUZUKI, T.; KUROKAWA, H.; SHIMIZU, T.; Acc. Chem. Res. 39 (2006) 1, 37-43; Inst. Multidisc. Res. Adv. Mater., Tohoku Univ., Aoba, Sendai 980, Japan; Eng.) -Lindner 19-265

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. PAS is an acronym formed from the names of the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM). The heme cofactor in its PAS domain can act as a sensor of the cellular redox state that regulates the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase activity. The crystal structures of its heme-containing PAS domain have helped clarify how the heme redox-dependent structural changes initiate intramolecular signal transduction. Here, we review recent findings on the structure-function relationships of EcDOS.

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. For this experiment, ProteoChip, generally suitable for antibody immobilization, was used as solid substrate. In this report, we confirm the antibody immobilization ability of ProteoChip and specific binding to the F(c) region of the antibody. Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PAS domain were investigated on the solid surface. Ec DOS immobilized via anti-(His)6-Tag mAb maintained interactions with the PAS fragment, in contrast to directly immobilized Ec DOS in the absence of anti-(His)6-Tag mAb. Heme-redox-sensitive interactions between Ec DOS and the PAS fragment were additionally detected using anti-(His)6-Tag mAb as a mediator. Our results collectively suggest that the immobilization method using anti-Tag antibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures and functions on solid surfaces.

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. the standard hydrogen electrode in the wild-type enzyme to 95 mV and 114 mV in the Ala and Asn mutants, respectively. Additionally, the auto-oxidation rate of Ec DOS PAS was significantly increased from 0.0053 to 0.051 and 0.033 min )1 , respectively. Interestingly, the catalytic activities of the Asp40 mutants were abolished completely. Thus, Asp40 appears to play a critical role in the electronic structure of the haem iron and redox-dependent catalytic control of the PDE domain. In this report, we discuss the mechanism of catalytic control of Ec DOS, based on the physico-chemical characteristics of the Asp40 mutants.Keywords: auto-oxidation; haem axial ligand; haem sensor; phosphodiesterase; redox potential.Haem-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a haem sensor enzyme composed of two functional domains: an N-terminal haem-bound sensor domain and a C-terminal PDE catalytic domain [1,2]. Catalysis by this enzyme is regulated by the haem redox state in that PDE is functional in the Fe(II) haembound enzyme, but not the Fe(III) haem-bound enzyme [2,3]. The crystal structure of the haem-bound domain revealed a typical PAS structure [4,5]. PAS proteins display a characteristic three-dimensional structure with a glove-like fold consisting of five juxtaposed b-sheets and flanking a-helices [6][7][8][9]. The characteristic three-dimensional structure of the PAS domain is commonly used for discussing the signal transduction mechanism of numerical signal transducing enzymes [6][7][8][9]. The crystal structures of both the Fe(II) and Fe(III) forms of the isolated haem-bound PAS domain (Ec DOS PAS) disclose that haem axial ligand switching from His77/ hydroxide anion to the His77/Met95 ligand pair occurs upon haem reduction. Moreover, haem ligand switching induces conformational changes in the FG loop region and movement of two subunits. These structural changes may play critical roles in catalytic regulation of the PDE domain [4].Structures of the haem-bound PAS domain have been reported under various conditions, and structure-function relationships are well documented [8][9][10][11][12][13][14]. FixL is an oxygen sensor enzyme with a haem-bound PAS domain. Specifically, O 2 association/dissociation to/from the haem switches off/on catalysis [8,9]. Global structural changes at the haem distal side are induced upon O 2 binding to FixL, and these changes contribute significantly to intramolecular signal transduction [9][10][11]. For Ec DOS, site-directed mutations at Met95, the axial ligand at the distal side in the Fe(II) complex, induced significant changes in the redox p...