Structure- and Function-based Characterization of a New Phosphoglycolate Phosphatase from Thermoplasma acidophilum

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel β-sheet with strand order S10, S9, S8, S1, S2 and a small β-hairpin, strands S3 and S4. This central sheet is flanked by a set of three α-helices on one side and two helices on the other. The smaller domain is composed of an open faced β-sandwich represented by three antiparallel β-strands, S5, S6, and S7, flanked by two oppositely oriented α-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl- ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase. The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel β-sheet with strand order S10, S9, S8, S1, S2 and a small β-hairpin, strands S3 and S4. This central sheet is flanked by a set of three α-helices on one side and two helices on the other. The smaller domain is composed of an open faced β-sandwich represented by three antiparallel β-strands, S5, S6, and S7, flanked by two oppositely oriented α-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl- ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase. Acid hydrolases are ubiquitous enzymes that catalyze a diverse array of reactions such as phosphate hydrolysis and phosphoryl transfer. Acid hydrolases belong to the haloacid dehalogenase (HAD) 1The abbreviations used are: HAD, haloacid dehalogenase; pNPP, p-nitrophenyl phosphate; PSP, phosphoserine phosphatase; SBC, Structural Biology Center; SeMet, selenomethionine; TA, Thermoplasma acidophilum; DALI, distance matrix alignment. superfamily, which includes l-2-haloacid dehalogenase, epoxide hydrolase, phosphoserine phosphatase, phosphomannomutase, phosphoglycolate phosphatase, and P-type ATPase, all of which utilize a nucleophilic aspartate in their phosphoryl transfer reaction. HAD superfamily proteins with different catalytic activities have low sequence identity, less than 14%; however, they are characterized by the following three conserved sequence motifs: motif I, DX(D/T/Y)X(T/V)(L/V); motif II, (S/T); and motif III, K(G/S)(D/S)XXX(D/N) (1.Aravind L. Galperin M.Y. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 2.Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Motif I is the most conserved; only subtle variations in this motif are observed for different functional classes of the HAD superfamily. In motif I, the first Asp, which is conserved in all HAD family members, is the functional nucleophile (2.Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). This nucleophile becomes phosphorylated and is directly involved in the phosphoryl transfer reaction. The second Asp is found in all phosphotransferases and phosphatases but is replaced by a Thr in P-type ATPases and by a Tyr in dehalogenases (2.Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). This Asp residue partakes both in the acid-base catalysis reaction and in the phosphorylation of the first Asp of motif I. Motif II contains a conserved serine or threonine that hydrogen bonds to the phosphoryl oxygen of the substrate and helps to orient it for the nucleophilic attack (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The conserved residues, except lysine, from motif III interact with an active site divalent metal (in most cases Mg2+) and are essential for activity (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 4.Collet J.F. Stroobant V. Van Schaftingen E. J. Biol. Chem. 1999; 274: 33985-33990Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The conserved lysine interacts with the phosphoaspartate intermediate, which results in the stabilization of its phosphorylated state. This lysine, although intimately involved in the catalytic mechanism, is located far away in primary sequence from the conserved sequence DXXXD in motif III and is located on a different secondary structure element in the protein. Crystal structures of proteins from the HAD superfamily include 2-haloacid dehalogenase, phosphonoacetaldehyde hydrolase, Ca2+-ATPase of sarcoplasmic reticulum, and phosphoserine phosphatase (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 5.Regni C. Tipton P.A. Beamer L.J. Structure (Camb.). 2002; 10: 269-279Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 6.Morais M.C. Zhang W. Baker A.S. Zhang G. Dunaway-Mariano D. Allen K.N. Biochemistry. 2000; 39: 10385-10396Crossref PubMed Scopus (125) Google Scholar, 7.Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1613) Google Scholar, 8.Hisano T. Hata Y. Fujii T. Liu J.Q. Kurihara T. Esaki N. Soda K. J. Biol. Chem. 1996; 271: 20322-20330Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). These proteins all share a conserved α/β-domain classified as a hydrolase fold, which is similar to the Rossmann fold. Residues from the conserved sequence motifs are also conserved at the tertiary level as shown from the crystal structures of the proteins for this superfamily. TA0175 was selected for structural studies as part of our structural proteomic project because it did not have a structural homologue and was annotated as “unknown” in the genome data base. Multiple sequence analysis using the non-redundant amino acid sequence data base identified more than 50 similar proteins with sequence identities ranging from 75 to 22%, none of which had a known biological function. However, a subset of these proteins were annotated as “putative hydrolase,” and they all contained the three conserved motifs observed for the HAD superfamily, which suggested that TA0175 is a potential acid hydrolase. As stated above, this family comprises a number of different enzymes; it is impossible to determine the catalytic function from sequence analysis because there is low sequence conservation among these different functional classes of proteins. Here we demonstrate how the three-dimensional crystal structure, when combined with sequence and biochemical analysis, led to the functional annotation of TA0175 as phosphoglycolate phosphatase. Cloning, Protein Expression, and Purification—The TA0175 gene (GenBank™ accession number gi 16081332) was cloned from genomic Thermoplasma acidophilum (TA) DNA, and its gene product was expressed, selenomethionine (SeMet)-labeled, and purified from a bacterial system using the nickel-nitrilotriacetic acid affinity procedure as described elsewhere for Methanobacterium thermoautotrophicum proteins (9.Christendat D. Saridakis V. Dharamsi A. Bochkarev A. Pai E.F. Arrowsmith C.H. Edwards A.M. J. Biol. Chem. 2000; 275: 24608-24612Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Screening for crystallization conditions was also performed as described elsewhere for M. thermoautotrophicum proteins (9.Christendat D. Saridakis V. Dharamsi A. Bochkarev A. Pai E.F. Arrowsmith C.H. Edwards A.M. J. Biol. Chem. 2000; 275: 24608-24612Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Crystallization experiments were conducted using the hanging drop vapor diffusion method at room temperature, 296 K. The final crystallization condition consisted of 22% polyethylene glycol 3350 as the precipitant, 0.2 m calcium chloride (CaCl2), and sodium HEPES at pH 7.5. The crystals chosen for x-ray data collection were flash frozen in this buffer containing 12% glycerol, which acted as cryoprotectant. The morphology of the single crystals is rods with maximum dimensions of 0.3 × 0.1 × 0.4 mm3. X-ray Diffraction and Structure Determination—Crystals of the TA0175 belong to the orthorhombic space group I222 with unit cell dimensions a = 88.2, b = 99.2, c = 113.8 Å, α = β = γ = 90°. Multiwavelength anomalous dispersion data were collected on SBC-2, a 3 × 3 charged coupled device detector built at Advanced Photon Source, to 1.7-Å resolution from a single crystal containing SeMet-labeled protein at three different wavelengths (peak, inflection, and high remote) near the selenium edge (Table I). The native data were collected to 1.4 Å and used for the later stages of the refinement of the protein structure. All diffraction data were collected at 100 K at the 19ID beamline of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory. The multiwavelength anomalous dispersion and native data were using the of W. PubMed Scopus Google Scholar). collection are presented in I. multiwavelength anomalous dispersion and by were using J. M. T. Biol. 1998; PubMed Scopus Google Scholar). The protein was using at Å A. R. Biol. 1999; PubMed Scopus Google Scholar). The refinement was the data collected at the of to Å, by of of and of the was using both and by The used in the refinement includes the with and of K. The was using the following a of at in the with distance in the of Å with In the later stages of the refinement were also which of and of The final were with the native data collected to 1.4 Å using the refinement procedure as The final is with the of All were using and and were with the M. A. PubMed Scopus Google Scholar). are in The J. Google Biol. Scopus Google and The Center for were used for of the of crystal and multiwavelength anomalous dispersion data collection = Å, b = Å, c = Å, α = β = γ = of in a of and TA0175 refinement of is the number of from of Protein Protein side in most in in is the number of in a Enzymatic activity with phosphate was in 50 HEPES buffer as described C.H. H. PubMed Scopus Google Scholar). phosphatase activity was as described by Biochem. PubMed Scopus Google except that was activity with phosphorylated was using reaction final containing 50 HEPES buffer 0.1 m 50 and of a at the reaction was by the of 0.2 of Biochem. PubMed Scopus Google and at room the at was were by using the kinetic = and kinetic were with the Acid sequence analysis of TA0175 with non-redundant amino acid in the protein data base identified more than 50 bacterial proteins that are annotated as or small than were classified as belonging to the HAD superfamily of All of these proteins share between and 22% of sequence with TA0175 with the three sequence motifs observed in the HAD superfamily conserved among these proteins sequence analysis of these proteins revealed that their primary were all to the data by genomic functional analysis had performed for this group of proteins. sequence analysis revealed that TA0175 to the of of proteins hydrolases and more to a The of the proteins with to TA0175 were with This analysis revealed conservation of motif I, is residue for this of hydrolases analysis revealed that purified TA0175 is a This is with our from this crystallization we observed two of TA0175 in The is by a which an and from to the base of this structural and is found in the of this at the The dimensions of TA0175 are × × Å The between the two is α the Structure is composed of two distinct domains, a larger the hydrolase fold, and a smaller domain. The core domain is composed of a centrally located five-stranded parallel β-sheet with strand order S10, S9, S8, S1, S2 and a small β-hairpin, strands S3 and S4. The β-sheet is flanked by three α-helices on the side and by two α-helices on the side of the the the and contains a a an This of secondary structure in the core the parallel S9, S8, S1, and and the helices the in the is similar to that observed for proteins belonging to the HAD superfamily, phosphoserine phosphatase and from (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, J.F. K. A. W. E. 2002; PubMed Scopus Google secondary is also observed for the core domain of a bacterial E. A.M. J. Biol. PubMed Scopus Google Scholar). The core domain is by the of a small α/β-domain The of the core domain from residue 50 to residue between strand and The is an open faced β-sandwich represented by three antiparallel β-strands, S5, S6, and S7, and two oppositely oriented α-helices, H3 and The first strand S5, and the second is by strands and S7, which a The analysis of a of this protein revealed a the active site of this protein is with and is as a the This high of in the active site is with observed for acid Structure analysis of TA0175 was by its with of proteins in the Protein using the The structural to TA0175 are PSP, phosphonoacetaldehyde hydrolase, from sarcoplasmic reticulum, l-2-haloacid dehalogenase, and epoxide hydrolase (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 5.Regni C. Tipton P.A. Beamer L.J. Structure (Camb.). 2002; 10: 269-279Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 6.Morais M.C. Zhang W. Baker A.S. Zhang G. Dunaway-Mariano D. Allen K.N. Biochemistry. 2000; 39: 10385-10396Crossref PubMed Scopus (125) Google Scholar, 7.Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1613) Google Scholar, 8.Hisano T. Hata Y. Fujii T. Liu J.Q. Kurihara T. Esaki N. Soda K. J. Biol. Chem. 1996; 271: 20322-20330Abstract Full Text Full Text PDF PubMed Scopus (139) Google (Table All of these proteins belong to the HAD their structures of an domain with an domain. The topology of the core domain proteins in this superfamily is The of the structure of TA0175 with that of revealed that the core α/β-domain with a of Å from the In from the three sequence motifs of structural to protein TA0175 with protein in a the core domain structural with the the cap domain no structural with of the different functional classes of HAD enzymes identified by In the cap domain is an open β-sandwich of three antiparallel flanked by two In PSP, the cap domain is a in phosphonoacetaldehyde hydrolase it is a These structural that the biological function of TA0175 from that of the proteins. known bacterial identified by the analysis share structural to of and catalyze phosphoryl transfer to an active site aspartate and are of bacterial interacts with as part of a reaction. has a single domain with an similar to the HAD superfamily. and also share active site similar to the active site aspartate of motif I from the HAD superfamily E. A.M. J. Biol. PubMed Scopus Google Scholar, D. J. Biol. 2000; PubMed Scopus Google Scholar). However, the cap domain is not seen in the it is that TA0175 to this of bacterial of both sequence analysis and structure of we have the of TA0175 and of its sequence to a subset of proteins in the HAD superfamily. The sequence analysis of this of proteins was performed by for the motif containing found in TA0175 and its sequence In the HAD superfamily, the first aspartate which is the nucleophile for this of is The second conserved aspartate in this which is in is found only in phosphotransferases and phosphatases and is replaced by a threonine in ATPases and by a in that TA0175 not have or dehalogenase The threonine found at in the motif is conserved in all enzymes except and The residue found at in the motif is replaced by a serine in interacts with the phosphate oxygen of the the of the to attack by the nucleophilic aspartate P.A. Sci. A. 2001; PubMed Scopus Google Scholar, W. Kim R. Jancarik J. Yokota H. Kim S.H. J. Biol. 2002; PubMed Scopus Google Scholar). these are with TA0175 is to of the following acid a a phosphoserine phosphatase, a phosphatase, a a P-type ATPase, or a haloacid sequence and structural analysis suggested that the biological function of TA0175 is different from a number of phosphatases from the of potential functional PSP, phosphonoacetaldehyde hydrolase, ATPase, l-2-haloacid dehalogenase, epoxide hydrolase, and results from the structural and sequence analysis are with TA0175 belonging to one of the of hydrolases phosphatase, a phosphatase, a phosphomannomutase, or a phosphoglycolate or that TA0175 belong to a of acid hydrolases TA0175 was for biological activity of the enzymes by for the of phosphate in the of their found that TA0175 catalyzed only the dephosphorylation of phosphoglycolate and kinetic similar to the enzyme phosphoglycolate phosphatase Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The pH divalent metal chloride and kinetic of TA0175 were with of phosphoglycolate phosphatase from and from these studies are with TA0175 as a phosphoglycolate phosphatase (Table and of kinetic for TA0175 with of eukaryotic phosphoglycolate × × × phosphoglycolate × cell phosphoglycolate × Biochem. PubMed Scopus Google in a activity of TA0175 with phosphoglycolate was kinetic was also the of TA0175 to catalyze the of phosphate from the two phosphatase and As enzyme TA0175 catalyzed the of phosphate from both and however, the catalytic behavior of this enzyme is substrate pNPP, are observed but with catalytic and catalytic with the than with phosphoglycolate as the enzyme catalytic properties are with its catalytic by less than However, we observed a of a with a = 0.2 in This is with the crystal structure of the which revealed different distinct of the the protein. phosphatase from both and is by ions Biochem. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar). The phosphoglycolate phosphatase activity of TA0175 was by and with the is known to phosphoglycolate phosphatase K. K. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of in than inhibition of the phosphoglycolate phosphatase activity of TA0175 Biochem. PubMed Scopus Google have shown that Cl- ions are at low to and are competitive with to phosphoglycolate at analysis with TA0175 is also with the at low chloride to 50 the enzyme was but with chloride its activity was and at phosphoglycolate phosphatase inhibition In the results from our kinetic of TA0175 the that this protein is a phosphoglycolate of and as for TA0175 of to the reaction results in of However, is shown to have the than inhibition was observed with ions in the reaction behavior was also observed with phosphoglycolate phosphatase from of Cl- ions in TA0175 a low of Cl- to is shown to the activity of the enzyme However, at Cl- ions The the competitive inhibition of high of Cl- These at the which is of competitive inhibition Cl- ions were shown to have a similar on phosphoglycolate phosphatase from of of the three-dimensional structure of with that of this identified T. acidophilum phosphoglycolate phosphatase revealed in their active The identified in motifs I, II, and III of the HAD superfamily were all located in the active site of this phosphoglycolate phosphatase. These active site with active site of The functional of a number of these conserved have for by and J.F. Stroobant V. Van Schaftingen E. J. Biol. Chem. 1999; 274: 33985-33990Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In crystal structures of with Mg2+, and with have all to the of the HAD superfamily of proteins (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 7.Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1613) Google Scholar, W. Kim R. Jancarik J. Yokota H. Kim S.H. J. Biol. 2002; PubMed Scopus Google Scholar). of and crystal structure of TA0175 revealed ions The protein was in the of its for Mg2+, the divalent metal as seen from our kinetic analysis, is not The active site of which was identified by the was located between and in the three-dimensional structure. The active are located on of the and from to a active In one we identified two ions that interact with conserved seen in motifs I, II, and The first of these ions interacts with and two The and of this is to the of the found in proteins such as and (3.Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). second is found Å away from the first in the structure. This is to the of and three The in a Å away from the two in the and only two and the of with the protein This not with the protein in the we observed with three in These are in by hydrogen with and the of residue is that these the substrate of this enzyme because in enzymes are known to substrate from Crystal crystal structure of TA0175 from the three conserved sequence motifs with a which most replaced the in the protein. a similar with has observed for acid and the of these in the catalytic has described elsewhere W. Kim R. Jancarik J. Yokota H. Kim S.H. J. Biol. 2002; PubMed Scopus Google Scholar). that the observed for acid such as and P-type ATPase, is also conserved for phosphoglycolate phosphatase on conserved structural conserved active site residues, conserved and conserved sequence motifs in this HAD superfamily P.A. Sci. A. 2001; PubMed Scopus Google Scholar). In or divalent ions to orient the nucleophilic which the of The of the is by two with helps to the the of the on the and of the phosphate with and the of this for nucleophilic The set of the for TA0175 to catalyze the hydrolysis of The hydrolysis of phosphoglycolate is by the nucleophilic attack by on the This results in the of the and a phosphorylated enzyme with the of is that the of with the phosphate is also to this phosphorylated The active site a to which this an active enzyme for of phosphate This phosphorylated Asp was observed in this and elsewhere J.F. Stroobant V. Van Schaftingen E. J. Biol. Chem. 1999; 274: 33985-33990Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). have shown that at low pH a phosphorylated is however, at high pH this phosphorylated is (Table from purified TA0175 different TA0175 in a In of substrate the crystal structure of TA0175 that the of of the protein are not between its two we observed that the in this in different in observed three with the and that they the of the biological In the the is and the active This is by between and is that the of most the of substrate and product to and from the active and the between and this and of in enzyme phosphoglycolate phosphatase has from from and from In it is involved in the dephosphorylation of which is when with oxygen phosphatase is in the its is a of and K. K. H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In phosphoglycolate is by and has shown to the of which is a of the oxygen affinity of Biochem. PubMed Scopus Google Scholar). In the of phosphoglycolate phosphatase is to of phosphoglycolate and it to by the However, in the of phosphoglycolate phosphatase is not a functional phosphoglycolate phosphatase did not an A. A. E. E. 1999; PubMed Scopus Google Scholar). the gene this enzyme is found in the as the gene gene product is involved in the of in the by or These are to the and for to function T. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The enzyme involved in of the has to studies to conducted to determine the identified phosphoglycolate phosphatase a in in all of the Structural Biology Center at Argonne National and the Center for Structural for in

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