DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli
DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli
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DOI：
10.1128/jb.187.19.6678-6682.2005
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发表时间：
2005-10
影响因子：
3.2
通讯作者：
Tokiko Yoshimura-Suzuki;I. Sagami;N. Yokota;H. Kurokawa;Toru Shimizu
中科院分区：
文献类型：
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作者：
Tokiko Yoshimura-Suzuki;I. Sagami;N. Yokota;H. Kurokawa;Toru Shimizu
大肠杆菌血红素调节磷酸二酯酶（DOSEc）在体外催化环腺苷酸（cAMP）水解，受血红素氧化还原状态的调节。氧化还原状态的变化导致酶的三维结构发生变化，然后将其传递到功能域以打开或关闭催化。因为DOSEc最初是从E. coli基因组DNA中表达，但目前尚不清楚它是否真的在野生型E.杆菌此外，使用cAMP作为底物的DOSEc的转换数仅为0.15 min-1，这对于生理学相关酶来说相对较低。在本研究中，我们首次证明了DOSEc基因和蛋白在野生型大肠杆菌中表达。大肠杆菌，特别是在有氧条件下。我们还开发了DOSEc基因敲除菌株（Δdos）。有趣的是，dos的敲除在有氧条件下引起细胞内cAMP的过量积累（比野生型菌株高26倍），而在厌氧条件下没有观察到cAMP的积累。我们还发现突变细胞和野生型菌株之间的细胞形态和生长速率的差异。通过引入dos的表达质粒，部分补充了敲除菌株中的变化。因此，本研究表明，DOSEc的表达是根据环境O2的可用性在转录水平上进行调节，细胞中cAMP的浓度受DOSEc表达的调节。
ABSTRACT Heme-regulated phosphodiesterase from Escherichia coli (DOSEc) 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 DOSEc 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 DOSEc 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 DOSEc gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOSEc gene knockout strain (Δdos). 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 DOSEc is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOSEc expression.