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Discovery of a cAMP Deaminase That Quenches Cyclic AMP-Dependent Regulation

Goble AM, Feng Y, Raushel FM, Cronan JE. (2013) ACS Chem Biol 8, 2622-9. PMCID: PMC3880142

The biological role of a newly discovered AH superfamily member, termed CadD, was deciphered in this study the AH Bridging Project and Microbiology Core. This effort builds on the emphasis by the EFI to go beyond biochemical characterization and understand the underlying physiology behind an enzymatic activity. This work provides a prime example of the interesting biology that can be uncovered when additional in vivo experiments are coupled to in vitro characterization.

ABSTRACT

An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3',5'-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3',5'-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 105 M-1 s-1 and has no activity on adenosine, adenine, or 5'-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes.

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2013 Goble 1 Abstract Image

2013 Goble 2 Fig 1.  Sequence similarity network created using Cytoscape (www.cytoscape.org) of COG1816 from the amidohydrolase superfamily. Each node in the network represents a single sequence, and each edge (depicted as lines) represents the pairwise connection between two sequences at a BLAST E-value of better than 1 × 10–70. Lengths of edges are not significant, except for tightly clustered groups, which are more closely related than sequences with only a few connections. Groups 1, 2, 5, 6, and 8 contain the adenosine deaminase enzymes including the E. coli adenosine deaminase in Group 5. In Group 3, the sequences depicted in yellow are adenine deaminases whereas the pink sequences represent cytokinin deaminase enzymes. Group 18 (blue) contains CadD, the enzyme studied in this investigation. All proteins of Group 18 are from Leptospira strains.

2013 Goble 3 Fig 2. CadD purification, identification, and enzymatic properties. Panels A and B. SDS-polyacrylamide gel analyses of purified CadD protein with detection by either Coomassie Blue staining or by Western blotting with an antihexahistidine antibody. Panel C. Mass spectral identification of the recombinant CadD protein. The peptide fragments matching the database sequence are given in bold and underlined type. Panel D. The CadD reaction. Panel E. CadD deamination of cAMP. The CadD concentration was 5 nM. Panel E. Inhibition of CadD by cPuMP (inset). Inhibition by cPuMP of CadD activity in 25 mM potassium HEPES (pH 7.5). The solid line represents the fit of the data to eq 2

2013 Goble 4 Fig 3.CadD expression mimics adenylate cyclase deficiency. Panel A. Growth of E. coli wild type strain MG1655 on various carbon sources in the presence or absence of CadD expression. Glucose does not require cAMP for its utilization whereas cAMP is required for expression of the genes encoding the proteins required to utilize the other sugars. IPTG (0.5 mM) was added for induction of CadD expression. The plates are sectored by plastic walls to prevent cross-feeding. Panels B–E. The cytosolic levels of cAMP in the presence or absence of CadD expression were determined in various strains. For comparison, panel B shows the effects of deleting cyaA, the gene encoding adenylate cyclase. * denotes p < 0.05 whereas ** denotes p < 0.01. Two E. coli K-12 backgrounds were used. Strain MC4100 was used in much of the early work on cAMP control, but it is unable to grow on lactose or arabinose due to deletion of the lacZYA and araBAD operons, respectively. Hence, the wild type strain MG1655 was used to assay response to these sugars. Strain MC4100 was used for the LacZ fusion studies because its lacZYA deletion eliminates background ß-galactosidase activity. The genomes of both strains have been fully sequenced and their cyaA and crp genes and flanking sequences are identical.(34)

2013 Goble 5 Fig 4. Effects of cAMP, cIMP, and CadD on DNA binding by CRP protein. Panels A and B show electrophoretic mobility assays done using 0.2 pmol of a dioxigenin-labeled fadD probe and CRP at the levels given in a total volume of 20 μL. In panel A, the effects of cIMP and cadD were assayed, whereas in panel B the ability of cIMP to displace cAMP from CRP and thereby prevent DNA binding was tested.

2013 Goble 6 Fig 5. Effects of CadD expression on transcription of cAMP–CRP dependent genes. Panel A. Alignment of the CRP binding sites. Panels B–D show the effects of CadD expression on transcription of lacZ and two fatty acid β-oxidation genes fabD and fabH. Panels E and F show the effect of CadD induction and exogenous cAMP addition on transcription of the fad genes. Expression of CadD and LacZ was induced by IPTG (0.5 mM) whereas the β-oxidation genes were induced by 5 mM oleic acid. * denotes p < 0.001 whereas ** denotes p < 0.0001. Strain MG1655 was assayed for lacZ whereas the fad strains were the chromosomal integrants described previously.

Reprinted with permission from ACS Chemical Biology.
Copyright © 2013, American Chemical Society