DOS _Ec , a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli

Abstract

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. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOS Ec using cAMP as a substrate is only 0.15 min ؊1 , which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOS Ec gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOS Ec gene knockout strain (⌬dos). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOS Ec is regulated according to environmental O 2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOS Ec expression.As described by , the heme-regulated phosphodiesterase from Escherichia coli (DOS Ec ) is composed of an N-terminal heme-bound PAS domain and a Cterminal phosphodiesterase domain. The phosphodiesterase activity of this enzyme hydrolyzes adenosine 3Ј,5Ј-cyclic monophosphate (cAMP) when the heme iron is in the ferrous (Fe 2ϩ ) but not in the ferric (Fe 3ϩ ) state (18). Changes in the redox state of the heme-bound iron are transduced to the enzyme's catalytic domain, thus regulating the catalytic activity (6,12,18,20,21,24). Crystallographic analysis has revealed that reduction of the heme iron induces a global conformational change in the FG loop within the heme-binding domain and causes the replacement of a heme-bound water with a side chain of . These profound structural changes in the PAS domain accompanied by the heme redox change are transmitted to the phosphodiesterase domain so that the heme redox state can act as an on/off switch for the enzyme. Thus, DOS Ec can be classified as a heme-based sensor.DOS Ec was originally identified in the E. coli genomic DNA sequence (6). Despite detailed biochemical and biophysical studies (6,12,18,20,21,24), it has been unclear whether the DOS Ec protein and gene (dos) are actually expressed in wildtype E. coli. In addition, the turnover number of DOS Ec using cAMP as a substrate is only 0.15 min Ϫ1 , which is relatively low for a physiological enzyme and brings into question whether it plays a role in cAMP regulation in vivo. Therefore, further studies are n...

Biological roles of cAMP: variations on a theme in the different kingdoms of life

“…In E. coli in addition to the class III PDE, cpdA (Imamura et al ., ), there is an atypical PDE regulated by heme, Ec direct oxygen sensor ( Ec DOS) (Sasakura et al ., ). Although cyclic diGMP is a much better substrate for Ec DOS than cAMP (Schmidt, Ryjenkov & Gomelsky, ), the activity on cAMP appears physiologically relevant, since an E. coli strain lacking DOS accumulates high levels of cAMP (Yoshimura‐Suzuki et al ., ).…”

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

“…Such redox-dependent structural changes appear to reflect the redox-dependent regulation in the cAMP phosphodiesterase activity of Ec DOS. Furthermore, knocking down the Ec DOS gene in E. coli causes changes in cell growth rate, cell morphology, and intracellular cAMP concentration, suggesting that Ec DOS plays an important role in modulating the cellular effects of cAMP (13).…”

“…In contrast, the EAL domain in Ec DOS appears to contain the catalytic domain, so that this domain retains high phosphodiesterase activity that rapidly converts c-diGMP into linear dinucleotide GMP (l-diGMP) (3,4). Furthermore, 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).…”

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole

“…DosP cleaves both c‐di‐GMP (Schmidt et al, ) and cAMP (Sasakura et al, ), although c‐di‐GMP is proposed as the physiological substrate due to higher activity towards c‐di‐GMP than cAMP (Schmidt et al, ). Nevertheless, DosP is important for maintaining cAMP levels (Yoshimura‐Suzuki et al, ), which are tightly regulated at low intracellular concentrations (Hantke et al, ). DosP is active as a tetramer and is strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor (Sasakura et al, ).…”