Discovery of a cAMP deaminase that quenches cyclic AMP-dependent regulation.

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 × 10(5) 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.

Deamination of 6-aminodeoxyfutalosine in menaquinone biosynthesis by distantly related enzymes.

Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10(5) M(-1) s(-1), Km values of 0.9-6.0 µM, and kcat values of 1.2-8.6 s(-1). Adenosine, 2'-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after ß-strand 1 to be partially responsible for the substrate specificity of Sav2595.

Discovery of a cytokinin deaminase.

An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins. Cytokinins are a common type of plant hormone and N-6-substituted adenines are also found as modifications to tRNA. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a k(cat)/K(m) of 1.2 × 10(7) M(-1) s(-1). Additional substrates include N-6-benzyl adenine, cis- and trans-zeatin, kinetin, O-6-methylguanine, N-6-butyladenine, N-6-methyladenine, N,N-dimethyladenine, 6-methoxypurine, 6-chloropurine, and 6-thiomethylpurine. This enzyme does not catalyze the deamination of adenine or adenosine. A comparative model of Patl2390 was computed using the three-dimensional crystal structure of Pa0148 (PDB code 3PAO ) as a structural template, and docking was used to refine the model to accommodate experimentally identified substrates. This is the first identification of an enzyme that will hydrolyze an N-6-substituted side chain larger than methylamine from adenine.

Pa0148 from Pseudomonas aeruginosa catalyzes the deamination of adenine.

Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with k(cat)/K(m) values that exceed 10(5) M(-1) s(-1). These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted (ß/α)(8) barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine, and hypoxanthine were also determined, thereby permitting identification of the residues responsible for coordinating the substrate and product.