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. The enhancement is also regulated by the heme redox state of the isolated PAS domain. In the present study, we used a newly developed protein microarray system to examine the relationship between catalytic activity and the interaction of full-length Ec DOS and the isolated PAS fragment. Adenosine 3',5'-cyclic monophosphate (cAMP), a substrate of the Ec DOS phosphodiesterase, was found to be indispensable for the interaction between Ec DOS and the PAS fragment, and two phosphodiesterase inhibitors, 3-isobutyl-methyl-xanthine and etazolate hydrochloride, hindered the interaction. In addition, an enzyme with a mutation in the putative cAMP-binding sites (H590 and H594) was unable to interact with Ec DOS and lacked enzymatic activity. These results strongly suggest a close relationship between Ec DOS phosphodiesterase activity and interaction with the isolated PAS fragment. Therefore, this study provides insights into the mechanism of how the isolated PAS domain activates Ec DOS, which has important implications for the general role of the isolated PAS domain in cells. Moreover, we found that multiple microscale analyses using the protein microarray system had several advantages over conventional affinity column methods, including the quantity of protein needed, the sensitivity, the variability of immobilized protein, and the time required for the experiment.
Sasakura
Yukie
Y
Bio-Medical Center, R&D Division, Nanotechnology Product Business Group, Hitachi High-Technologies Corporation, Hitachinaka, Ibaraki, 312-8504, Japan.
Kanda
Katsuhiro
K
Yoshimura-Suzuki
Tokiko
T
Matsui
Takuya
T
Fukuzono
Shinichi
S
Shimizu
Toru
T
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D056950:Period Circadian Proteins; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases; D040081:Protein Array Analysis / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D000069296:Type III Secretion Systems
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000409:Alanine; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D000242:Cyclic AMP; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D006639:Histidine; D009687:Nuclear Proteins; D056950:Period Circadian Proteins; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D000069296:Type III Secretion Systems; C497397:Arnt protein, mouse; C114344:PAS protein, E coli; C539040:PER protein, Drosophila; C089449:sim protein, Drosophila; C464427:dosP protein, E coli