Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m805996200
ISSN1083-351X
AutoresRicha Tyagi, Avinash R. Shenoy, Sandhya S. Visweswariah,
Tópico(s)Mechanisms of cancer metastasis
ResumoAmong the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2′,3′-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events. Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2′,3′-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events. With the advent of large scale genome sequencing efforts along with more sophisticated methods of genetic mapping, a number of loci have been identified that are associated with human disease. Intriguingly, many genes identified in these loci remain uncharacterized at the biochemical and functional level, and although current annotation can provide a general prediction of the function of some proteins, understanding finer aspects of their regulation require individual analysis at a single gene level. Chromosome 11p13-14 has been extensively studied because of its association with various tumors, among them lung and bladder cancers (1Shipman R. Schraml P. Colombi M. Raefle G. Ludwig C.U. Hum. Genet. 1993; 91: 455-458Crossref PubMed Scopus (34) Google Scholar, 2Bepler G. Garcia-Blanco M.A. Proc. Natl. Acad. Sci. U. S. 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Based on extensive physical and genetic mapping combined with clinical and cytogenetic data, high resolution integrated maps have been constructed for chromosome 11p13-14 region (13Compton D.A. Weil M.M. Jones C. Riccardi V.M. Strong L.C. Saunders G.F. Cell. 1988; 55: 827-836Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 14Fantes J.A. Oghene K. Boyle S. Danes S. Fletcher J.M. Bruford E.A. Williamson K. Seawright A. Schedl A. Hanson I. Zehetner G. Bhogal R. Lehrach H. Gregory S. Williams J. Little P.F. Sellar G.C. Hoovers J. Mannens M. Weissenbach J. Junien C. Vanheyningen V. Bickmore W.A. Genomics. 1995; 25: 447-461Crossref PubMed Scopus (60) Google Scholar, 15Junien C. McBride O.W. Cytogenet. Cell Genet. 1989; 51: 226-258Crossref PubMed Scopus (50) Google Scholar, 16Gessler M. Bruns G.A. Genomics. 1989; 5: 43-55Crossref PubMed Scopus (46) Google Scholar, 17Gessler M. Thomas G.H. Couillin P. Junien C. McGillivray B.C. Hayden M. Jaschek G. Bruns G.A. Am. J. Hum. 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Gene (Amst.). 1997; 194: 57-62Crossref PubMed Scopus (8) Google Scholar). Although the function of the 239FB protein remains unknown, the distinctive expression of the gene in the fetal brain and the presence of an "ancient conserved region" (21Schwartz F. Ota T. Gene (Amst.). 1997; 194: 57-62Crossref PubMed Scopus (8) Google Scholar) in this gene suggest its role in the development of the nervous system. In current databases (Genecards), the 239FB gene has been annotated as a metallophosphoesterase (MPE), 4The abbreviations used are: MPE, metallophosphoesterase; CthPnkp, C. thermocellum polynucleotide kinase-phosphatase; DLS, dynamic light scattering; SNP, single nucleotide polymorphism; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; PDE, phosphodiesterase.4The abbreviations used are: MPE, metallophosphoesterase; CthPnkp, C. thermocellum polynucleotide kinase-phosphatase; DLS, dynamic light scattering; SNP, single nucleotide polymorphism; GST, glutathione S-transferase; MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; PDE, phosphodiesterase. and a close ortholog of this protein, called 239AB, is expressed in the adult brain (21Schwartz F. Ota T. Gene (Amst.). 1997; 194: 57-62Crossref PubMed Scopus (8) Google Scholar). Neither protein has hitherto been characterized. Metallophosphoesterases are a large group of enzymes that catalyze a variety of diverse reactions. All members contain five blocks of residues and within these blocks are found 11 invariant residues, DXnGDXnGNH(E/D)XnHXnGHXH, and the structural motif conserved in metallophosphoesterases is the β-α-β-α-β-fold (22Hopfner K.P. Karcher A. Craig L. Woo T.T. Carney J.P. Tainer J.A. Cell. 2001; 105: 473-485Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 23Miller D.J. Shuvalova L. Evdokimova E. Savchenko A. Yakunin A.F. Anderson W.F. Protein Sci. 2007; 16: 1338-1348Crossref PubMed Scopus (23) Google Scholar, 24Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar). The superfamily of metallophosphoesterases is made up of two functionally and structurally well characterized groups, phosphomonoesterases and phosphodiesterases. Whereas phosphomonoesterases have been divided into protein Ser/Thr phosphatases, purple acid phosphatases, and 5′-nucleotidases, diesterases are categorized into phospholipases, 2′,3′-cyclic nucleotide phosphodiesterases, UDP 2′,3′-diacylglucosaminase, and Class III phosphodiesterases (25Ek-Rylander B. Bergman T. Andersson G. J. Bone Miner. Res. 1991; 6: 365-373Crossref PubMed Scopus (21) Google Scholar, 26Zimmermann H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (745) Google Scholar). The amino acid residues involved in coordinating the dimetal center are located at the carboxyl end of the parallel β-strands of the fold (23Miller D.J. Shuvalova L. Evdokimova E. Savchenko A. Yakunin A.F. Anderson W.F. Protein Sci. 2007; 16: 1338-1348Crossref PubMed Scopus (23) Google Scholar, 24Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar). Structural and biochemical analysis of these proteins have shown the metal requirements (Fe2+, Fe3+, Mn2+, Mg2+, Zn2+, Co2+, Ni2+) at the dimetal nuclear center for activity (27Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2134) Google Scholar, 28Koonin E.V. Mol. Microbiol. 1993; 8: 785-786Crossref PubMed Scopus (15) Google Scholar, 29Uppenberg J. Lindqvist F. Svensson C. Ek-Rylander B. Andersson G. J. Mol. Biol. 1999; 290: 201-211Crossref PubMed Scopus (106) Google Scholar, 30Zhang J. Zhang Z. Brew K. Lee E.Y. Biochemistry. 1996; 35: 6276-6282Crossref PubMed Scopus (91) Google Scholar). Despite the large number of MPEs that have been biochemically characterized, the function of these proteins in the organism remains enigmatic, and no natural substrate has been identified for many of them (23Miller D.J. Shuvalova L. Evdokimova E. Savchenko A. Yakunin A.F. Anderson W.F. Protein Sci. 2007; 16: 1338-1348Crossref PubMed Scopus (23) Google Scholar, 31Vogel A. Schilling O. Niecke M. Bettmer J. Meyer-Klaucke W. J. Biol. Chem. 2002; 277: 29078-29085Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 32Chen S. Yakunin A.F. Kuznetsova E. Busso D. Pufan R. Proudfoot M. Kim R. Kim S.H. J. Biol. Chem. 2004; 279: 31854-31862Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Moreover, it is not readily possible to classify the MPEs as either monoesterases or diesterases by inspection of the sequences of the proteins. Recently, we have biochemically and structurally characterized the Rv0805 gene from Mycobacterium tuberculosis, which to date is the only identifiable cyclic nucleotide phosphodiesterase in M. tuberculosis (24Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar, 33Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar). The Rv0805 protein is a Class III cyclic nucleotide phosphodiesterase, which are enzymes found almost exclusively in bacteria. The catalytic core of Rv0805 is distantly related to the calcineurin-like phosphatases, and a unique substrate binding pocket was identified by computational docking (24Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar). During our attempts to identify orthologs of this gene in different organisms, we were surprised to see that a number of genes similar to Rv0805 protein could be identified in eukaryotes and among them were the 239FB and 239AB genes. In the present study we have cloned the full-length cDNAs of the 239FB and 239AB genes from rat brain, and using extensive biochemical and mutational analysis, we have shown that these proteins are indeed metallophosphoesterases. A single nucleotide polymorphism (SNP) seen in the FB gene inactivates the protein, and because the 239AB protein can hydrolyze 2′,3′-cyclic nucleotide phosphodiesters, this is the first report of a functional Class III PDE in eukaryotes. All nucleotides as well as the colorimetric substrates p-nitrophenyl phosphate, bis(p-nitrophenyl) phosphate, p-nitrophenyl thymidine 5′-monophosphate, p-nitrophenyl phenylphosphonate, and p-nitrophenylphosphorylcholine were obtained from Sigma-Aldrich. Malachite green reagent was purchased from MP Biomedicals. Bioinformatic Analysis for Identification of ICC/CpdA-like Proteins—The metallophosphoesterase superfamily encompasses a large number of protein sub-families that have in common a core seven-residue bimetal binding active site that catalyzes the hydrolysis of various phospho-, mono-, and diester substrates. The Pfam data base of protein families groups all these proteins by a single hidden Markov model (PF00149) built on 332 seed sequences. A cyclic nucleotide phosphodiesterase from Escherichia coli (product of the ICC/CpdA gene) is a metallophosphoesterase, and the Rv0805 is similar to this protein (33Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar). As we aimed to identify ICC-related sequences, we relied on the established subclassifications of highly related proteins in the Clusters of Orthologous Groups data base (www.ncbi.nlm.nih.gov/COG). Eight proteins of 332 Pfam seed sequences matched to Cog2129 (ICC-related) using RPS-BLAST. These sequences were aligned using PROBCONS and the alignment used to generate a new hidden Markov model that would identify only ICC-related phosphodiesterases and prevent all other metallophosphoesterases from being enlisted in the desired inventory of ICC-like proteins. A HMMER (34Eddy S.R. Bioinformatics. 1998; 14: 755-763Crossref PubMed Scopus (3958) Google Scholar) search of the non-redundant data base of proteins revealed the presence of ∼130 eukaryotic ICC-like proteins (supplemental Fig. 1 and Table I) among a total of 505 hits and identified two human proteins, 239AB and 239FB. Expression and Purification of 239FB—RNA was isolated from fetal (20 days of gestation) and adult rat brain using TRI reagent (Sigma) as per the manufacturer's protocol and subjected to first strand cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (Promega). The rat 239FB full-length coding region was amplified using 239FBfwdMun1 and 239FBrvs primers on the fetal brain cDNA (supplemental Table II). PCR was performed with DeepVent DNA polymerase (New England Biolabs), and the purified PCR product was digested with MunI and XhoI and cloned into the EcoRI and XhoI sites of pBKS II vector (Invitrogen). The sequence of the cloned fragment was identical to accession number NM_198778. For expression of the protein, a clone was generated using a forward primer containing an Nco1 site and cloned as an NcoI-XhoI fragment into pPROExHT-C to generate plasmid pPRO-239FB1-294. This allowed expression in E. coli of the 239FB protein with a hexahistidine tag. The D65A, H67A, H67R, D86A, E89A, N117A, H118A, H213A, G252H, H254A mutations were generated on the pPRO-239FB1-294 plasmid using a single oligonucleotide-based mutagenesis protocol (35Shenoy A.R. Visweswariah S.S. Anal. Biochem. 2003; 319: 335-336Crossref PubMed Scopus (93) Google Scholar). The sequences of the oligonucleotides used for mutagenesis are provided in supplemental Table II, and all inserts were sequenced to confirm the presence of only the desired mutations (Macrogen). Proteins were expressed in the E. coli BL21DE3 strain on induction using isopropyl β-d-1-thiogalactopyranoside (500 μm) for 20 h at 16 °C. Cells were lysed by sonication in buffer containing 50 mm Tris/HCl (pH 8.2), 5 mm 2-mercaptoethanol, 100 mm NaCl, 10% glycerol, 1 mm benzamidine, and 2 mm phenylmethylsulfonyl fluoride followed by centrifugation at 30,000 × g. The supernatant was interacted with nickel-nitrilotriacetic acid beads (Qiagen), and the matrix was washed with a similar buffer. This was followed by washes with a buffer containing 100 mm Tris/HCl (pH 8.2), 5 mm 2-mercaptoethanol, 500 mm NaCl, and 20 mm imidazole, and elution in the buffer containing 100 mm Tris/HCl (pH 8.2), 5 mm 2-mercaptoethanol, 50 mm NaCl, and 300 mm imidazole. Purified protein was desalted into buffer containing 50 mm Tris/HCl (pH 8.2), 5 mm 2-mercaptoethanol, 50 mm NaCl, and 10% glycerol and stored in aliquots at -70 °C until further use. For expression of 239FB fused to an N-terminal GST tag, a BamHI-XhoI fragment from the pBKS II-239FB plasmid was cloned in to the BamHI and XhoI sites of pGEX-6P3 (GE Healthcare) to generate plasmid, pGEX-6P3-239FB. Gel Filtration and Dynamic Light Scattering—Gel filtration was carried out in buffer containing 50 mm Tris/HCl, 5 mm 2-mercaptoethanol, 50 mm NaCl, and 10% glycerol at pH 8.8 and 4 °C at a flow rate of 200 μl/min using a Superose 12 column and an AKTA fast protein liquid chromatography system (GE Healthcare). The column was calibrated using commercially available gel filtration standards containing thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa). Protein estimation of peaks containing 239FB protein was performed by the modified Bradford method (36Zor T. Selinger Z. Anal. Biochem. 1996; 236: 302-308Crossref PubMed Scopus (862) Google Scholar). Dynamic Light Scattering experiments were performed on a DynaPro Molecular Sizing Instrument (Protein Solutions). DLS measures fluctuations in the intensity of light scattered by a macromolecular solution which can be related to its hydrodynamic radius (Rh). Samples of 239FB protein were analyzed directly after gel filtration at concentrations of ∼400 μg/ml. Protein solutions were centrifuged three times at 16,000 × g for 15 min and immediately loaded into a quartz cuvette before measurement. Several measurements were taken at 277 K and analyzed using DYNAMICS Version 3.30 software (Protein Solutions). Data collection times of 10 s were used in all cases, for a minimum of 15 acquisitions. Western Blot Analysis of 239FB—Purified 239FB protein was injected into rabbits to raise an antibody to the protein by standard procedures as described earlier (33Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar). Rat fetal brain tissues were homogenized in homogenization buffer containing 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, 1 mm dithiothreitol, 100 mm NaCl, 100 mm sodium fluoride, 10 mm sodium pyrophosphate, 80 μm β-glycerol phosphate, 1 mm benzamidine, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor, and 100 μm sodium orthovanadate, after which samples were centrifuged at 12,000 g for 1 h at 4 °C. Aliquots were stored at -70 °C. Total brain proteins (25 μg) were fractionated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blotting was carried out using the immunoglobulin fraction (3 μg/ml) prepared from the immune serum as described earlier (33Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar). Blots were subsequently probed with Hsp70 antibody (Stressgen; 200 ng/ml) to normalize protein levels in the individual lanes. Metallophosphoesterase Enzymatic Assays—Assays for various activities (phosphatase, phosphodiesterase, nuclease, phospholipase) were performed in a triple buffer system (MES, HEPES, diethanolamine, 50 mm (pH 9.0) (37Newman J. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 610-612Crossref PubMed Scopus (106) Google Scholar), 5 mm 2-mercaptoethanol, and 10 mm NaCl in the presence of 10 mm concentrations of the specified substrate and Mn2+ as the metal cofactor. The triple buffer system allowed a change in a wide range of pH without altering the ionic composition of the buffer. Assays were stopped by the addition of 10 μl of 200 mm NaOH, and absorbance was monitored at 405 nm. The amount of p-nitrophenol formed was estimated based on its molar extinction coefficient of 18,450 m-1 cm-1. For kinetic analysis assays were performed with the same buffer system as described earlier with 239FB protein and different concentrations of substrates and metals (Ni2+ and Mn2+) as indicated. Each measurement was performed in triplicate for two different enzyme preparations. Hydrolysis of single-stranded M13 DNA or double-stranded pUC19 DNA or RNA (yeast RNA type II) was assayed in 20 μl of reaction volume in buffer containing 50 mm HEPES (pH 8.0), 10 mm NaCl, 1 mm dithiothreitol, and 5 mm MnCl2 at 37 °C for 120 min in the presence of 1 μg of 239FB. The reactions were stopped by the addition of 6× loading dye, and the reaction products were analyzed on 0.8% agarose, 1× Tris acetate, EDTA gels containing 1 μg/ml ethidium bromide. Cyclic Nucleotide Phosphodiesterase Assays—Hydrolysis of cyclic mononucleotides (2′,3′-cAMP, 3′,5′-cAMP, and 3′,5′-cGMP) was analyzed in reaction mixtures in the three buffer system (50 mm MES, HEPES, diethanolamine (pH 9.0)), 5 mm 2-mercaptoethanol, 10 mm NaCl, and 5 mm MnCl2 with different concentrations of the specified substrate in the presence of 1 μg of 239FB and 0.1 unit of calf intestine phosphatase. The reaction was stopped after 90 min of incubation at 37 °C by the addition of 50 μl of a malachite green solution, prepared as described previously (38Harder K.W. Owen P. Wong L.K. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (182) Google Scholar, 39Baykov A.A. Evtushenko O.A. Avaeva S.M. Anal. Biochem. 1988; 171: 266-270Crossref PubMed Scopus (677) Google Scholar). Absorbance was measured at 620 nm to detect the released inorganic phosphate, and the amount of released phosphate was estimated by interpolating the value to a standard curve generated with inorganic phosphate. For analysis of the cyclic nucleotide PDE assay by HPLC, 1 μg of 239FB was incubated for 120 min with 10 mm of the specified substrate as well as metal cofactor in the buffer system mentioned above in a volume of 25 μl. The reaction was terminated by the addition of 50 μl of 20 mm (NH4)H2PO4 (pH 6.2). Aliquots from assays were applied to a Supelcosil LC-8DB (25 cm × 4.6 mm, 5 μm) HPLC column equilibrated with 20 mm (NH4)H2PO4 (pH 6.2) at a flow rate of 1 ml/min on an Agilent Technologies HPLC system. The column was calibrated with a mixture of nucleotides containing 2′,3′-cAMP, 3′,5′-cAMP, 3′-AMP, 5′-AMP, and 2′-AMP. Cloning and Expression of Rat 239AB—The full-length coding region of rat 239AB was amplified using 239ABfwd and 239ABrvs primers and Deep Vent polymerase (supplemental Table II) from cDNA prepared from the adult rat brain. Single 3′-A overhangs were added to the PCR product using TaqDNA polymerase and then ligated in to pGEMT Easy vector (Promega). The clone was sequenced and subcloned as a BamHI and NotI fragment into the BamHI and NotI sites of pGEX-5X2 (GE Healthcare) to generate plasmid pGEX-5X2-239AB, encoding 239AB fused to an N-terminal GST tag. GST-FB and GST-AB proteins were expressed in E. coli BL21DE3 strain after induction with isopropyl β-d-1-thiogalactopyranoside (500 μm) for 20 h at 16 °C. Cells were lysed by sonication in a buffer containing 50 mm Tris/HCl (pH 8.2), 1 mm dithiothreitol, 100 mm NaCl, 2 mm EDTA, 10% glycerol, 1 mm benzamidine, and 2 mm phenylmethylsulfonyl fluoride followed by centrifugation at 30,000 × g. The supernatant was interacted with GSH beads, and the matrix was washed with a similar buffer. This was followed by washes with a buffer containing 50 mm Tris/HCl (pH 8.2), 1 mm dithiothreitol, 100 mm NaCl, 2 mm EDTA, and 0.1% Triton-X. Subsequently, beads were washed with a buffer containing 50 mm Tris/HCl (pH 8.2), 1 mm dithiothreitol, 100 mm NaCl, 2 mm EDTA, and 10% glycerol, and stored at 4 °C. Protein was estimated by addition of Bradford reagent to the beads suspension. Identification of 239FB as an Ortholog of the Rv0805 Gene—During our earlier studies on cyclic nucleotide metabolism in bacteria, we identified and characterized both structurally and biochemically the Rv0805 3′,5′-cAMP PDE from M. tuberculosis (24Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar, 33Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar). This protein hydrolyzed cAMP in vitro and in vivo and, therefore, may contribute to cAMP homeostatis in mycobacteria. Given the unique features of Rv0805 and the availability of structural information, we sought to identify similar proteins in other genomes. The MPE family encompasses several subfamilies of homologous proteins grouped based on their substrate specificities. Here we have narrowed down our searches to identify as many members possible of the subfamily that could hydrolyze 3′,5′-cAMP and -cGMP. Using a combined RPS-BLAST and hidden Markov model approach, we found that although not all genomes contain Rv0805-like proteins, these genes nonetheless seem to have a wide phyletic distribution. The majority of proteins identified were of bacterial or archaebacterial in origin (supplemental Table I). This was expected because eukaryotic cyclic nucleotide phosphodiesterases belong to a phylogenetically unrelated group of proteins, the class I PDEs (40Conti M. Beavo J. Annu. Rev. Biochem. 2007; 76: 481-511Crossref PubMed Scopus (930) Google Scholar). However, we were intrigued to find some orthologs in eukaryotes, including mammals (Fig. 1; supplemental Table I and Fig. 1). Of the 505 genes identified, 287 were found in bacteria, 82 in archaebacteria, 135 in eukaryotes, and a single gene in a virus. The majority of sequence diversity in the MPE domain was found in archaebacteria followed by proteobacteria and actinobacteria, among the eubacterial lineages. We, therefore, selected the most diverse members among the bacterial genes to construct a representative phylogenetic tree retaining Rv0805 and a few other eukaryotic sequences to highlight their relative placement and bring out the diversification within several bacterial and archaeal clades. We were surprised to notice the close relation between Rv0805 and the mammalian proteins (Fig. 1), and the availability of biochemical and structural information on Rv0805 prompte
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