Crystal structure of trehalose-6-phosphate phosphatase-related protein: biochemical and biological implications

We report here the crystal structure of a trehalose-6-phosphate phosphatase-related protein (T6PP) from Thermoplasma acidophilum, TA1209, determined by the dual-wavelength anomalous diffraction (DAD) method. T6PP is a member of the haloacid dehalogenase (HAD) superfamily with significant sequence homology with trehalose-6-phosphate phosphatase, phosphoserine phosphatase, P-type ATPases and other members of the family. T6PP possesses a core domain of known alpha/beta-hydrolase fold, characteristic of the HAD family, and a cap domain, with a tertiary fold consisting of a four-stranded beta-sheet with two alpha-helices on one side of the sheet. An active-site magnesium ion and a glycerol molecule bound at the interface between the two domains provide insight into the mode of substrate binding by T6PP. A trehalose-6-phosphate molecule modeled into a cage formed by the two domains makes favorable interactions with the protein molecule. We have confirmed that T6PP is a trehalose phosphatase from amino acid sequence, three-dimensional structure, and biochemical assays.     

Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 A resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6(F)-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 x 40 x 35 A(3), and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded beta-sheet surrounded by four alpha-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.     

Structure of recombinant Haemophilus influenzae e (P4) acid phosphatase reveals a new member of the haloacid dehalogenase superfamily

Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class C phosphatase. P4 has a two-domain architecture consisting of a core alpha/beta domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.     

Crystal structure of the probable haloacid dehalogenase PH0459 from Pyrococcus horikoshii OT3

PH0459, from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, is a probable haloacid dehalogenase with a molecular mass of 26,725 Da. Here, we report the 2.0 A crystal structure of PH0459 (PDB ID: 1X42) determined by the multiwavelength anomalous dispersion method. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by six alpha-helices and three 3(10)-helices. One disulfide bond, Cys186-Cys212, forms a bridge between an alpha-helix and a 3(10)-helix, although PH0459 seems to be an intracellular protein. The subdomain inserted into the core domain has a four-helix bundle structure. The crystal packing suggests that PH0459 exists as a monomer. A structural homology search revealed that PH0459 resembles the l-2-haloacid dehalogenases l-DEX YL from Pseudomonas sp. YL and DhlB from Xanthobacter autotrophicus GJ10. A comparison of the active sites suggested that PH0459 probably has haloacid dehalogenase activity, but its substrate specificity may be different. In addition, the disulfide bond in PH0459 probably facilitates the structural stabilization of the neighboring region in the monomeric form, although the corresponding regions in l-DEX YL and DhlB may be stabilized by dimerization. Since heat-stable dehalogenases can be used for the detoxification of halogenated aliphatic compounds, PH0459 will be a useful target for biotechnological research.     

The Crystal Structure of Arabidopsis VSP1 Reveals the Plant Class C-Like Phosphatase Structure of the DDDD Superfamily of Phosphohydrolases

Arabidopsis thaliana vegetative storage proteins, VSP1 and VSP2, are acid phosphatases and belong to the haloacid dehalogenase (HAD) superfamily. In addition to their potential nutrient storage function, they were thought to be involved in plant defense and flower development. To gain insights into the architecture of the protein and obtain clues about its function, we have tested their substrate specificity and solved the structure of VSP1. The acid phosphatase activities of these two enzymes require divalent metal such as magnesium ion. Conversely, the activity of these two enzymes is inhibited by vanadate and molybdate, but is resistant to inorganic phosphate. Both VSP1 and VSP2 did not exhibit remarkable activities to any physiological substrates tested. In the current study, we presented the crystal structure of recombinant VSP1 at 1.8 Å resolution via the selenomethionine single-wavelength anomalous diffraction (SAD). Specifically, an α-helical cap domain on the top of the α/β core domain is found to be involved in dimerization. In addition, despite of the low sequence similarity between VSP1 and other HAD enzymes, the core domain of VSP1 containing conserved active site and catalytic machinery displays a classic haloacid dehalogenase fold. Furthermore, we found that VSP1 is distinguished from bacterial class C acid phosphatase P4 by several structural features. To our knowledge, this is the first study to reveal the crystal structure of plant vegetative storage proteins.     

Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities

Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg²⁺-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5′-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin.     

The Structural Domains of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Cooperate in Substrate Hydrolysis: 3D Structure and Enzymatic Mechanism

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It colonizes different tissues by the utilization of diverse mechanisms. One of these may involve the breakdown of the host cell membrane through the sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PchP). The action of hemolytic phospholipase C on phosphatidylcholine produces phosphorylcholine, which is hydrolyzed to choline (Cho) and inorganic phosphate by PchP. The available biochemical data on this enzyme demonstrate the involvement of two Cho-binding sites in the catalytic cycle and in enzyme regulation. The crystal structure of P. aeruginosa PchP has been determined. It folds into three structural domains. The first domain harbors all the residues involved in catalysis and is well conserved among the haloacid dehalogenase superfamily of proteins. The second domain is characteristic of PchP and is involved in the recognition of the Cho moiety of the substrate. The third domain stabilizes the relative position of the other two. Fortuitously, the crystal structure of PchP captures molecules of Bistris (2‐[bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol) at the active site and at an additional site. This represents two catalytically relevant complexes with just one or two inhibitory Bistris molecules and provides the basis of the PchP function and regulation. Site‐directed mutagenesis along with biochemical experiments corroborates the structural observations and demonstrates the interplay between different sites for Cho recognition and inhibition. The structural comparison of PchP with other phosphatases of the haloacid dehalogenase family provides a three‐dimensional picture of the conserved catalytic cycle and the structural basis for the recognition of the diverse substrate molecules.     

HAD superfamily phosphotransferase substrate diversification: structure and function analysis of HAD subclass IIB sugar phosphatase BT4131

The BT4131 gene from the bacterium Bacteroides thetaiotaomicron VPI-5482 has been cloned and overexpressed in Escherichia coli. The protein, a member of the haloalkanoate dehalogenase superfamily (subfamily IIB), was purified to homogeneity, and its X-ray crystal structure was determined to1.9 A resolution using the molecular replacement phasing method. BT4131 was shown by an extensive substrate screen to be a broad-range sugar phosphate phosphatase. On the basis of substrate specificity and gene context, the physiological function of BT4131 in chitin metabolism has been tentatively assigned. Comparison of the BT4131 structure alpha/beta cap domain structure with those of other type IIB enzymes (phosphoglycolate phosphatase, trehalose-6-phosphate phosphatase, and proteins of unknown function known as PDB entries , , and ) identified two conserved loops (BT4131 residues 172-182 and 118-130) in the alphabetabeta(alphabetaalphabeta)alphabetabeta type caps and one conserved loop in the alphabetabetaalphabetabeta type caps, which contribute residues for contact with the substrate leaving group. In BT4131, the two loops contribute one polar and two nonpolar residues to encase the displaced sugar. This finding is consistent with the lax specificity BT4131 has for the ring size and stereochemistry of the sugar phosphate. In contrast, substrate docking showed that the high-specificity phosphoglycolate phosphatase (PDB entry ) uses a single substrate specificity loop to position three polar residues for interaction with the glycolate leaving group. We show how active site "solvent cages" derived from analysis of the structures of the type IIB HAD phosphatases could be used in conjunction with the identity of the residues stationed along the cap domain substrate specificity loops, as a means of substrate identification.     

A C-Terminal Phosphatase Module Conserved in Vertebrate CMP-Sialic Acid Synthetases Provides a Tetramerization Interface for the Physiologically Active Enzyme

The biosynthesis of sialic acid-containing glycoconjugates is crucial for the development of vertebrate life. Cytidine monophosphate-sialic acid synthetase (CSS) catalyzes the metabolic activation of sialic acids. In vertebrates, the enzyme is chimeric, with the N-terminal domain harboring the synthetase activity. The function of the highly conserved C-terminal domain (CSS-CT) is unknown. To shed light on its biological function, we solved the X-ray structure of murine CSS-CT to 1.9 Å resolution. CSS-CT is a stable shamrock-like tetramer that superimposes well with phosphatases of the haloacid dehalogenase superfamily. However, a region found exclusively in vertebrate CSS-CT appears to block the active-site entrance. Accordingly, no phosphatase activity was observed in vitro, which points toward a nonenzymatic function of CSS-CT. A computational three-dimensional model of full-length CSS, in combination with in vitro oligomerization studies, provides evidence that CSS-CT serves as a platform for the quaternary organization governing the kinetic properties of the physiologically active enzyme as demonstrated in kinetic studies.     

MDP-1 is a new and distinct member of the haloacid dehalogenase family of aspartate-dependent phosphohydrolases

MDP-1 is a eukaryotic magnesium-dependent acid phosphatase with little sequence homology to previously characterized phosphatases. The presence of a conserved motif (Asp-X-Asp-X-Thr) in the N terminus of MDP-1 suggested a relationship to the haloacid dehalogenase (HAD) superfamily, which contains a number of magnesium-dependent acid phosphatases. These phosphatases utilize an aspartate nucleophile and contain a number of conserved active-site residues and hydrophobic patches, which can be plausibly aligned with conserved residues in MDP-1. Seven site-specific point mutants of MDP-1 were produced by modifying the catalytic aspartate, serine, and lysine residues to asparagine or glutamate, alanine, and arginine, respectively. The activity of these mutants confirms the assignment of MDP-1 as a member of the HAD superfamily. Detailed comparison of the sequence of the 15 MDP-1 sequences from various organisms with other HAD superfamily sequences suggests that MDP-1 is not closely related to any particular member of the superfamily. The crystal structures of several HAD family enzymes identify a domain proximal to the active site responsible for important interactions with low molecular weight substrates. The absence of this domain or any other that might perform the same function in MDP-1 suggests an "open" active site capable of interactions with large substrates such as proteins. This suggestion was experimentally confirmed by demonstration that MDP-1 is competent to catalyze the dephosphorylation of tyrosine-phosphorylated proteins.     

Sequence‐ and activity‐based screening of microbial genomes for novel dehalogenases

Dehalogenases are environmentally important enzymes that detoxify organohalogens by cleaving their carbon-halogen bonds. Many microbial genomes harbour enzyme families containing dehalogenases, but a sequence-based identification of genuine dehalogenases with high confidence is challenging because of the low sequence conservation among these enzymes. Furthermore, these protein families harbour a rich diversity of other enzymes including esterases and phosphatases. Reliable sequence determinants are necessary to harness genome sequencing-efforts for accelerating the discovery of novel dehalogenases with improved or modified activities. In an attempt to extract dehalogenase sequence fingerprints, 103 uncharacterized potential dehalogenase candidates belonging to the α/β hydrolase (ABH) and haloacid dehalogenase-like hydrolase (HAD) superfamilies were screened for dehalogenase, esterase and phosphatase activity. In this first biochemical screen, 1 haloalkane dehalogenase, 1 fluoroacetate dehalogenase and 5 l -2-haloacid dehalogenases were found (success rate 7%), as well as 19 esterases and 31 phosphatases. Using this functional data, we refined the sequence-based dehalogenase selection criteria and applied them to a second functional screen, which identified novel dehalogenase activity in 13 out of only 24 proteins (54%), increasing the success rate eightfold. Four new l -2-haloacid dehalogenases from the HAD superfamily were found to hydrolyse fluoroacetate, an activity never previously ascribed to enzymes in this superfamily.     

Do metabolic HAD phosphatases moonlight as protein phosphatases?

Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.     

Structure of Francisella tularensis AcpA: prototype of a unique superfamily of acid phosphatases and phospholipases C

AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-A resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded beta-sheet flanked by three alpha-helices on either side, with the active site located above the carboxyl-terminal edge of the beta-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases.     

Cloning of the pyridoxine 5'-phosphate phosphatase gene (pdxP) and vitamin B6 production in pdxP recombinant Sinorhizobium meliloti

A novel gene (pdxP) encoding a pyridoxine 5'-phosphate (PNP) phosphatase involved in the last step of pyridoxine biosynthesis was cloned from Sinorhizobium meliloti IFO 14782 on the basis of the peptide sequences of the natural enzyme. The pdxP gene is an open reading frame (708 bp) encoding 235 amino acid residues with a calculated molecular weight of 26,466. From its deduced amino acid sequence, it was predicted that the enzyme belongs to the haloacid dehalogenase superfamily. Transformants of Escherichia coli and S. meliloti by pdxP gene expression plasmids showed stimulated PNP phosphatase activities. When pdxP was overexpressed together with the PNP synthase gene (pdxJ) in S. meliloti, the recombinant strain produced 149 mg/l of pyridoxine, 46% and 16% higher than the host strain and the pdxJ recombinant of S. meliloti respectively.     

Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics

Cofilin is a key regulator of actin cytoskeletal dynamics whose activity is controlled by phosphorylation of a single serine residue. We report the biochemical isolation of chronophin (CIN), a unique cofilin-activating phosphatase of the haloacid dehalogenase (HAD) superfamily. CIN directly dephosphorylates cofilin with high specificity and colocalizes with cofilin in motile and dividing cells. Loss of CIN activity blocks phosphocycling of cofilin, stabilizes F-actin structures and causes massive cell division defects. Our findings identify a physiological phospho-serine protein substrate for a mammalian HAD-type phosphatase and demonstrate that CIN is an important novel regulator of cofilin-mediated actin reorganization.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Comparative modelling of human PHOSPHO1 reveals a new group of phosphatases within the haloacid dehalogenase superfamily

PHOSPHO1 is a recently identified phosphatase whose expression is upregulated in mineralizing cells and is implicated in the generation of inorganic phosphate for matrix mineralization, a process central to skeletal development. The enzyme is a member of the haloacid dehalogenase (HAD) superfamily of magnesium-dependent hydrolases. However, the natural substrate(s) is as yet unidentified and to date no structural information is known. We have identified homologous proteins in a number of species and have modelled human PHOSPHO1 based upon the crystal structure of phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The model includes the catalytic Mg(2+) atom bound via three conserved Asp residues (Asp32, Asp34 and Asp203); O-ligands are also provided by a phosphate anion and two water molecules. Additional residues involved in PSP-catalysed hydrolysis are conserved and are located nearby, suggesting both enzymes share a similar reaction mechanism. In PHOSPHO1, none of the PSP residues that confer the enzyme's substrate specificity (Arg56, Glu20, Met43 and Phe49) are conserved. Instead, we propose that two fully conserved Asp residues (Asp43 and Asp123), not present in PSPs contribute to substrate specificity in PHOSPHO1. Our findings show that PHOSPHO1 is not a member of the subfamily of PSPs but belongs to a novel, closely related enzyme group within the HAD superfamily.     

The X-ray crystallographic structure and activity analysis of a Pseudomonas-specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst.     

YbiV from Escherichia coli K12 is a HAD phosphatase

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized YbiV as a HAD phosphatase. The crystal structure of YbiV reveals a two-domain protein, one with the characteristic HAD hydrolase fold, the other an inserted alpha/beta fold. In an effort to understand the mechanism, we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3), which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate-binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate YbiV may be a sugar phosphatase, which is supported by biochemical assays that measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.