Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase

Rao, Feng; Pasunooti, Swathi; Ng, Yinglu; Zhuo, Weichao; Lim, Lishi; Liu, Angeline Weixian; Liang, Zhao‐Xun

doi:10.1016/j.ab.2009.03.031

Cited by 88 publications

(84 citation statements)

References 23 publications

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“…A modified diguanylate cyclase (tDGC) was purified and used to generate c-di-GMP enzymatically from GTP as described (32). Briefly, 1 mM GTP was added every hour to 5 μM protein in reaction buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 20 mM MgCl 2 ) in a total reaction volume of 10 mL for approximately 8 hours.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of differential ligand recognition by two classes of bis-(3′-5′)-cyclic dimeric guanosine monophosphate-binding riboswitches

Smith

Shanahan

Moore

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

106

195

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The bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbone is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.

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Section: Methodsmentioning

confidence: 99%

Structural basis of differential ligand recognition by two classes of bis-(3′-5′)-cyclic dimeric guanosine monophosphate-binding riboswitches

Smith

Shanahan

Moore

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

106

195

View full text Add to dashboard Cite

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“…Radioactively labeled c-di-GMP was enzymatically synthesized from [␣-32 P]GTP using the TM1788 DGC from T. maritima (tDGC), which was expressed and purified as previously described (37). Reaction mixtures incubated for 3 h at 30°C were boiled for 5 min and centrifuged to remove the precipitated tDGC, and the supernatant was applied to a 10K Ultrafree 0.5-ml filter (Millipore) to remove any leftover protein.…”

Section: Methodsmentioning

confidence: 99%

PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation

Chao

Rakshe

Leff

et al. 2013

Journal of Bacteriology

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Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (ϳ10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

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“…c-di-GMP was synthesized enzymatically using a thermophilic diguanylate cyclase protein as described previously (21,22).…”

Section: Methodsmentioning

confidence: 99%

Binding of Cyclic Diguanylate in the Non-catalytic EAL Domain of FimX Induces a Long-range Conformational Change

Chuah

Dong

et al. 2011

Journal of Biological Chemistry

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FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (ϳca. 70 Å ) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism.

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Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase

Cited by 88 publications

References 23 publications

Structural basis of differential ligand recognition by two classes of bis-(3′-5′)-cyclic dimeric guanosine monophosphate-binding riboswitches

Structural basis of differential ligand recognition by two classes of bis-(3′-5′)-cyclic dimeric guanosine monophosphate-binding riboswitches

PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation

Binding of Cyclic Diguanylate in the Non-catalytic EAL Domain of FimX Induces a Long-range Conformational Change

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