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Journal of Bacteriology, August 2009, p. 4722-4731, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00327-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase ^,

Feng Rao, Yaning Qi, Hui Shan Chong, Masayo Kotaka, Bin Li, Jinming Li, Julien Lescar, Kai Tang, and Zhao-Xun Liang^*

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551

Received 10 March 2009/ Accepted 11 April 2009

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/)₈ barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K_m) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K_m correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg²⁺ ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.

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* Corresponding author. Mailing address: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551. Phone: 65 63167866. Fax: 65 67913856. E-mail: zxliang{at}ntu.edu.sg

Published ahead of print on 17 April 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, August 2009, p. 4722-4731, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00327-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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Rationalizing the Evolution of EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterases

Ute Römling
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This article has been cited by other articles:

• Romling, U. (2009). Rationalizing the Evolution of EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterases. J. Bacteriol. 191: 4697-4700 [Full Text]