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Prospecting for unannotated enzymes: discovery of a 3',5'-nucleotide bisphosphate phosphatase within the amidohydrolase superfamily.

Cummings JA, Vetting M, Ghodge SV, Xu C, Hillerich B, Seidel RD, Almo SC, Raushel FM (2014) Biochemistry 53, 591-600. PMCID: PMC3985815

In a less traditional sequence of events, structural homology and Thermofluor-positive binding hits lead the Structure Core to the successful co-crystallization of a functionally uncharacterized protein (Cv1693 from Chromobacterium violaceum) belonging to the Amidohydrolase (AH) Superfamily. This new structural information then led the AH Bridging Project to the discovery of Cv1693's activity as a 3',5'-nucleotide bisphosphate phosphatase. Sequence Similarity Networks were then used in a retrospective manner to examine a subset of the AH superfamily, confirm the physiological substrate of Cv1693, and provisionally annotate 710 AH proteins. This study illuminates a unique and successful arrangement of EFI tool implementations.


In bacteria, 3',5'-adenosine bisphosphate (pAp) is generated from 3'-phosphoadenosine 5'-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5'-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn(2+) ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s(-1) and 1.4 × 10(6) M(-1) s(-1), respectively. The enzyme is promiscuous and is able to hydrolyze other 3',5'-bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2'-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes.

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Abstract Image

Figure 1: Sequence network map of cog0613 at an E value cutoff of 10–60 created using Cytoscape ( Each node (sphere) represents a protein sequence, while each edge (line) represents those sequence pairs that are more closely related than the arbitrary E value cutoff (10–60). The available crystal structures are shown as diamonds, and their respective PDB entries are given. The enzyme studied in this work, Cv1693 from C. violaceum ATCC 12472, and its orthologs are colored blue. Enzyme sequences colored yellow are those that have been annotated as trpH, while the single orange node represents cyclic phosphate hydrolase from E. lenta DSM 2243 (Elen0235), the only other enzymatic reaction known and demonstrated from cog0613.

Figure 2: Schematic of the secondary structure of Cv1693. The α- and β-metal sites, which are a common feature among members of the PHP family and some amidohydrolase enzymes, are shown as orange spheres, while the γ-metal site, unique to the PHP family members, is shown as a blue sphere. The strands and helices that constitute the distorted TIM- or (β/α)7-barrel are colored green and gray, respectively. The long β-strand-3/4 is colored purple, while the antiparallel β-strand 5, unique to members of cog0613 among AHS members, is highlighted with a red border. The insertion element consisting of α-helices is colored blue, and the positions of the enzyme residues seen to interact with the bound 5′-AMP are shown as red lines.

Figure 3: Stereo ribbon diagram of Cv1693. Helicies are shown as blue tubes and strands as orange arrows. Three bound manganese ions are shown as maroon spheres, and bound inorganic phosphate and adenosine monophosphate are shown as sticks, colored by atom type. The insertion sequence comprises helices α5a and α5b.

Figure 4: Fo – Fc electron density kick map contoured at 2.5σ. Interactions of the bound inorganic phosphate with the three manganese ions are shown as dashed lines.

Figure 5: Stereo diagram illustrating the ligands to the three bound manganese ions. Residues colored by atom type with the adenosine monophosphate with yellow carbons and protein atoms with white carbons. Interactions between protein atoms and the manganese atoms are shown as dashed lines.

Figure 6: Interactions of AMP with protein residues, the inorganic phosphate, and the manganese ion are shown as dashed lines.

Figure 7: Interactions of AMP with protein residues, the inorganic phosphate, and the manganese ion are shown as dashed lines.

Figure 8: Interactions of AMP with Bad1165. Residues are colored by atom type, and interactions of AMP with Bad1165 are shown as dashed lines. There is a water molecule that bridges Feα and Feβ.

Figure 9: Primary sequence alignment of proteins from cog0613: Cv1693, pAp phosphatase from C. violaceum (PDB entries 2YB1 and 2YB4); b1266, TrpH from Es. coli; Bad1165, enzyme of unknown function from B. adolescentis (PDB entries 3E0F and 3O0F); and Elen0235, cyclic phosphate dihydrolase from E. lenta. Residues binding the metal cofactors at the active site are colored red. Residues seen interacting with the bound 5′-AMP and inorganic phosphate in the crystal structure of Cv1693 are highlighted in yellow. Thr-135 is highlighted in blue. β-Sheets that constitute the (β/α)7-barrel are highlighted in gray.

Reprinted with permission from Biochemistry.
© 2011 American Chemical Society.