Critical Role of the Heme Axial Ligand, Met95, in Locking Catalysis of the Phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Atsunari Tanaka,Toru Shimizu,Hiroto Takahashi

doi:10.1074/jbc.m701920200

Abstract

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.

Highlights

JULY 20, 2007 VOLUME 282 NUMBER 29 development, virulence, antibiotic formation, and biofilm formation in bacteria growth and factor-stimulated proliferation in human colon cancer cells [1,2,3,4,5,6,7,8,9,10]
E. coli with a knock-out of the Ec DOS gene display a morphology and growth rate different from those of the wild-type strain, suggesting that the enzyme is important for cell development and/or proliferation [13]
That the phosphodiesterase activity of full-length, dithionite-reduced Ec DOS is up-regulated upon binding of either O2 minded protein); SmFixL, an oxygen sensor heme protein from Sinorhizobium meliloti; CooA, CO-sensing heme-bound transcriptional regulator from Rhodospirillum rubrum; DTT, dithiothreitol; Calf intestine alkaline phosphatase (CIAP), calf intestine alkaline phosphatase; NOC9, 6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-Nmethyl-1-hexanamine; PDE, phosphodiesterase

Summary

Introduction

JULY 20, 2007 VOLUME 282 NUMBER 29 development, virulence, antibiotic formation, and biofilm formation in bacteria growth and factor-stimulated proliferation in human colon cancer cells [1,2,3,4,5,6,7,8,9,10]. The optical absorption spectra of these Arg97 mutants in the absence or presence of CO and NO were essentially the same as for the wild-type protein, suggesting that the coordination structure (6-coordinated low-spin form) and the heme environment were not substantially changed by the Arg97 mutations.

Results

Conclusion