Crystal structure of the catalytic fragment of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), a member of the 2H phosphoesterase superfamily, is firmly bound to brain white matter and found mainly in the central nervous system of vertebrates, and it catalyzes the hydrolysis of 2',3'-cyclic nucleotide to produce 2'-nucleotide. Recent studies on CNP-knockout mice have revealed that the absence of CNP causes axonal swelling and neuronal degeneration. Here, the crystal structure of the catalytic fragment (CF) of human CNP (hCNP-CF) is solved at 1.8A resolution. It is an alpha+beta type structure consisting of three alpha-helices and nine beta-strands. The structural core of the molecule is comprised of two topologically equivalent three-stranded antiparallel beta-sheets that are related by a pseudo 2-fold symmetry. Each beta-sheet contains an H-X-T-X motif, which is strictly conserved among members of the 2H phosphoesterase superfamily. The phosphate ion is bound to the side-chains of His and Thr from each of the two motifs. Structural comparison of hCNP-CF with plant 1',2'-cyclic nucleotide phosphodiesterase (CPDase) and bacterial 2'-5' RNA ligase reveals that the H-X-T-X motifs are structurally conserved among these enzymes, but the surface properties of the active site are quite different among the enzymes, reflecting the differences in their substrates. On the basis of the present crystal structure of the hCNP-CF/phosphate complex, the available structure of the CPDase/cyclic-nucleotide analogue complex, and the recent functional studies of rat CNP-CF, we propose a possible substrate-binding mode and catalytic mechanism of CNP, which employs the nucleophilic water molecule activated by His310. The proposed mechanism is basically equivalent to the second step of the well-accepted reaction mechanism of RNase A. Since the overall structure of hCNP-CF differs considerably from that of RNase A, it is likely that the similar active sites with two catalytic histidine residues in these enzymes arose through convergent evolution.     

Saccharomyces cerevisiae Ngl3p is an active 3'-5' exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases

Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes.     

Conserved sequence pattern in a wide variety of phosphoesterases.

A unique sequence pattern, designated the GD/GNH signature, was shown to be conserved in a wide variety of phosphoesterases. The enzymes containing this signature cleave phosphoester bonds in such different substrates as (1) phosphoserine and phosphothreonine in polypeptides; (2) bis(5'-nucleosidyl)-tetraphosphates; (3) nucleoside 5' phosphates; (4) 2',3'-cyclic nucleotide phosphates; (5) polynucleotides; (6) 2'-5' phosphodiesters in RNA (intron) lariats; (7) sphingomyelin; and (7) various phosphomonoesters. Two conserved acidic amino acid residues and a conserved histidine residue may be directly involved in phosphoester bond cleavage.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

An end-healing enzyme from Clostridium thermocellum with 5' kinase, 2',3' phosphatase, and adenylyltransferase activities

We identify and characterize an end-healing enzyme, CthPnkp, from Clostridium thermocellum that catalyzes the phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and the dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp also catalyzes an autoadenylylation reaction via a polynucleotide ligase-type mechanism. These characteristics are consistent with a role in end-healing during RNA or DNA repair. CthPnkp is a homodimer of an 870-amino-acid polypeptide composed of three catalytic domains: an N-terminal module that resembles the polynucleotide kinase domain of bacteriophage T4 Pnkp, a central metal-dependent phosphoesterase module, and a C-terminal module that resembles the nucleotidyl transferase domain of polynucleotide ligases. The distinctive feature of CthPnkp vis-à-vis known RNA repair enzymes is that its 3' end modification component belongs to the calcineurin-type phosphatase superfamily. It contains putative counterparts of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. As with lambda phosphatase, the 2',3' cAMP phosphatase activity of CthPnkp is specifically dependent on nickel or manganese. We identify homologs of CthPnkp in other bacterial proteomes.     

DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history

A detailed analysis of the polbeta superfamily of nucleotidyltransferases was performed using computer methods for iterative database search, multiple alignment, motif analysis and structural modeling. Three previously uncharacterized families of predicted nucleotidyltransferases are described. One of these new families includes small proteins found in all archaea and some bacteria that appear to consist of the minimal nucleotidyltransferase domain and may resemble the ancestral state of this superfamily. Another new family that is specifically related to eukaryotic polyA polymerases is typified by yeast Trf4p and Trf5p proteins that are involved in chromatin remodeling. The TRF family is represented by multiple members in all eukaryotes and may be involved in yet unknown nucleotide polymerization reactions required for maintenance of chromatin structure. Another new family of bacterial and archaeal nucleotidyltransferases is predicted to function in signal transduction since, in addition to the nucleotidyltransferase domain, these proteins contain ligand-binding domains. It is further shown that the catalytic domain of gamma proteobacterial adenylyl cyclases is homologous to the polbeta superfamily nucleotidyltransferases which emphasizes the general trend for the origin of signal-transducing enzymes from those involved in replication, repair and RNA processing. Classification of the polbeta superfamily into distinct families and examination of their phyletic distribution suggests that the evolution of this type of nucleotidyltransferases may have included bursts of rapid divergence linked to the emergence of new functions as well as a number of horizontal gene transfer events.     

Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.     

The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit

The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The similarity of B-subunit sequences implies a common fold, but since the key catalytic and metal binding residues of the phosphoesterase domain are disrupted in the eukaryotic B-subunits, their common function has not been identified. To study the structure and activities of B-subunits in more detail, we expressed 13 different recombinant B-subunits in Escherichia coli. We found that the solubility of a protein could be predicted from the calculated GRAVY score. These scores were useful for the selection of proteins for successful expression. We optimized the expression and purification of Methanocaldococcus (Methanococcus) jannaschii DP1 of DNA polymerase D (MjaDP1) and show that the protein co-purifies with a thermostable nuclease activity. Truncation of the protein indicates that the N-terminus (aa 1-134) is not needed for catalysis. The C-terminal part of the protein containing both the calcineurin-like phosphoesterase domain and the OB-fold is sufficient for the nuclease activity.     

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.     

Structural evidence that brain cyclic nucleotide phosphodiesterase is a member of the 2H phosphodiesterase superfamily

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.     

Phosphoesterase domains associated with DNA polymerases of diverse origins.

Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases. The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase. The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis. The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity. Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases. The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs. A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization. As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward. The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes. In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members.     

Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.     

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous cofactor participating in numerous redox reactions. It is also a substrate for regulatory modifications of proteins and nucleic acids via the addition of ADP-ribose moieties or removal of acyl groups by transfer to ADP-ribose. In this study, we use in-depth sequence, structure and genomic context analysis to uncover new enzymes and substrate-binding proteins in NAD-utilizing metabolic and macromolecular modification systems. We predict that Escherichia coli YbiA and related families of domains from diverse bacteria, eukaryotes, large DNA viruses and single strand RNA viruses are previously unrecognized components of NAD-utilizing pathways that probably operate on ADP-ribose derivatives. Using contextual analysis we show that some of these proteins potentially act in RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we predict that another family of YbiA-related enzymes is likely to comprise a novel NAD-dependent ADP-ribosylation system for proteins, in conjunction with a previously unrecognized ADP-ribosyltransferase. A similar ADP-ribosyltransferase is also coupled with MACRO or ADP-ribosylglycohydrolase domain proteins in other related systems, suggesting that all these novel systems are likely to comprise pairs of ADP-ribosylation and ribosylglycohydrolase enzymes analogous to the DraG-DraT system, and a novel group of bacterial polymorphic toxins. We present evidence that some of these coupled ADP-ribosyltransferases/ribosylglycohydrolases are likely to regulate certain restriction modification enzymes in bacteria. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such as Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases and NEURL4-like ARTs, which might be involved in centrosomal assembly. We also infer a novel biosynthetic pathway that might be involved in the synthesis of a nicotinate-derived compound in conjunction with an asparagine synthetase and AMPylating peptide ligase. We use the data derived from this analysis to understand the origin and early evolutionary trajectories of key NAD-utilizing enzymes and present targets for future biochemical investigations.     

Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.     

3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism

3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II. The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs. Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme. The present study shows by computational analysis that the enzyme encoded by the E. coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases. They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH[E/D]-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature. Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis. In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases.     

Connectivity between Catalytic Landscapes of the Metallo-β-Lactamase Superfamily

The expansion of functions in an enzyme superfamily is thought to occur through recruitment of latent promiscuous functions within existing enzymes. Thus, the promiscuous activities of enzymes represent connections between different catalytic landscapes and provide an additional layer of evolutionary connectivity between functional families alongside their sequence and structural relationships. Functional connectivity has been observed between individual functional families; however, little is known about how catalytic landscapes are connected throughout a highly diverged superfamily. Here, we describe a superfamily-wide analysis of evolutionary and functional connectivity in the metallo-β-lactamase (MBL) superfamily. We investigated evolutionary connections between functional families and related evolutionary to functional connectivity; 24 enzymes from 15 distinct functional families were challenged against 10 catalytically distinct reactions. We revealed that enzymes of this superfamily are generally promiscuous, as each enzyme catalyzes on average 1.5 reactions in addition to its native one. Catalytic landscapes in the MBL superfamily overlap substantially; each reaction is connected on average to 3.7 other reactions whereas some connections appear to be unrelated to recent evolutionary events and occur between chemically distinct reactions. These findings support the idea that the highly distinct reactions in the MBL superfamily could have evolved from a common ancestor traversing a continuous network via promiscuous enzymes. Several functional connections (e.g., the lactonase/phosphotriesterase and phosphonatase/phosphodiesterase/arylsulfatase reactions) are also observed in structurally and evolutionary distinct superfamilies, suggesting that these catalytic landscapes are substantially connected. Our results show that new enzymatic functions could evolve rapidly from the current diversity of enzymes and range of promiscuous activities.     

RNA recognition by 3'-to-5' exonucleases: the substrate perspective

The 3'-to-5' exonucleolytic decay and processing of a variety of RNAs is an essential feature of RNA metabolism in all cells. The 3'-5' exonucleases, and in particular the exosome, are involved in a large number of pathways from 3' processing of rRNA, snRNA and snoRNA, to decay of mRNAs and mRNA surveillance. The potent enzymes performing these reactions are regulated to prevent processing of inappropriate substrates whilst mature RNA molecules exhibit several attributes that enable them to evade 3'-5' attack. How does an enzyme perform such selective activities on different substrates? The goal of this review is to provide an overview and perspective of available data on the underlying principles for the recognition of RNA substrates by 3'-to-5' exonucleases.     

Structural and biochemical characterization of a novel Mn2+-dependent phosphodiesterase encoded by the yfcE gene

Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis-p-nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 A resolution. YfcE has a beta-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique.