Structural and Functional Characterization of a Novel Phosphodiesterase from Methanococcus jannaschii

Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-Å resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two β-sheets flanked by α-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni2+ or Mn2+ produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5′-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni2+ and Mn2+ being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase. Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-Å resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two β-sheets flanked by α-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni2+ or Mn2+ produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5′-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni2+ and Mn2+ being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase. Several hundred genomes have been sequenced so far, but no function can be inferred from the gene sequence of about half of the genes. Structural information may provide new clues as to the molecular (biochemical and biophysical) functions of the proteins coded by these genes of unknown functions. An open reading frame from Methanococcus jannaschii, MJ0936 (GI 1499771), is annotated as a conserved hypothetical protein in the PEDANT sequence data base (1Frishman D. Kosykh D. Kastenmuller G. Kolesov G. Zubrzycki I. Gruber C. Geier B. Kaps A. Albermann K. Volz A. Wagner C. Fellenberg M. Heumann K. Mewes H.W. Nucleic Acids Res. 2003; 31: 207-211Google Scholar). A PSI-PHI BLAST search found 56 bacterial or archaeal homologs with E value less than 0.003. Some of the homologs are annotated, and they can be grouped into three categories: (i) (conserved) hypothetical protein; (ii) VPS29-like (human vacuolar protein-sorting protein) phosphoesterase-related protein; (iii) predicted/putative/probable phosphoesterase. Similarly, a Pfam (2Bateman A. Birney E. Durbin R. Eddy S.R. Howe K.L. Sonnhammer E.L. Nucleic Acids Res. 2000; 30: 276-280Google Scholar) search suggests that this gene may belong to a protein family of calcineurin-like phosphoesterase. This Pfam protein family contains a large range of phosphoesterases, including protein phosphoserine phosphatases, nucleotidases, sphingomyelin phosphodiesterases, 2′,3′-cyclic AMP phosphodiesterases, and nucleases such as bacterial SbcD or yeast MRE11, as well as the VPS29-like phosphoesterase-related proteins mentioned above. Presumed metal-chelating residues are found to be conserved for the proteins of this family. To determine the function of MJ0936 and its homologues, we have solved the crystal structure of this protein and performed a series of biochemical screenings for catalytic activity. Through a combination of structural and enzymatic analyses, we show that MJ0936 is a novel phosphodiesterase, and its three-dimensional structure disclosed key conserved residues involved in catalysis and metal binding. Cloning—Primers (Operon, Emeryville, CA) for PCR amplification contained an NdeI restriction site in the forward primer (5′-CATATGAAAATTGGGATAATGAGCG) and BamHI site in the reverse primer (5′-GGATCCTTACTCTAACACTATCTCCCTATACTCCTTCT). PCR was performed using Deep Vent Polymerase (New England Biolabs, Inc., Beverly, MA) and M. jannaschii genomic DNA. The PCR product was cloned into pCR-BluntII-TOPO vector (Invitrogen Corp., Carlsbad, CA) and the MJ0936 gene insert was confirmed by DNA sequencing. The amplified TOPO vector was restricted with NdeI and BamHI and the gene insert was purified by agarose gel electrophoresis extraction. This insert was ligated into pSKB3 (gift from Steve Burley, Rockefeller University, New York), digested with NdeI and BamHI, and transformed into DH5α. A plasmid containing the gene insert was confirmed and then transformed into BL21(DE3)pSJS1244 (3Kim R. Sandler S.J. Goldman S. Yokota H. Clark A.J. Kim S.H. Biotechnol. Lett. 1998; 20: 207-210Google Scholar). Protein Expression and Purification—His-tagged protein MJ0936 was expressed in Escherichia coli strain BL21(DE3)/pSJS1244 (3Kim R. Sandler S.J. Goldman S. Yokota H. Clark A.J. Kim S.H. Biotechnol. Lett. 1998; 20: 207-210Google Scholar) cells upon induction with 0.5 mm isopropyl-β-d-thiogalactopyranoside. Selenomethionyl protein was prepared according to the method of Doublie (4Doublie S. Methods Enzymol. 1997; 276: 523-529Google Scholar). Bacteria were lysed by sonication in Buffer A (50 mm Hepes, pH 7, 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml antipain, 1 μg/ml chymostatin, 0.5 μg/ml leupeptin, and 0.7 μg/ml pepstatin A) and cell debris was pelleted by centrifugation at 39,000 × g for 20 min in a Sorvall centrifuge. The lysate was then spun in a Beckman ultracentrifuge Ti45 rotor at 60,000 × g for 20 min at 4 °C to remove membrane proteins. The His-tagged MJ0936 protein was affinity purified from the soluble fraction using Talon metal affinity resin (Clontech, Palo Alto, CA) according to the procedure recommended by the manufacturer, elution was achieved with 600 mm imidazole. The eluted sample was dialyzed against 20 mm Tris, pH 7.5, 10 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA. The target protein was bound onto a 5-ml HiTrap Q column (Amersham Biosciences) and eluted with a 10-column volume linear gradient from 0.1 to 0.75 m NaCl in the same buffer. The purity of the expressed protein was determined by SDS-gel electrophoresis and the molecular weight was confirmed by electrospray mass spectrometry. The protein was concentrated in 20 mm Tris, pH 8.0, 1 mm EDTA, 1 mm dithiothreitol, 100 mm NaCl to 10 to 20 mg/ml. Enzymatic Assays—General enzymatic screens for various activities (phosphatase, phosphodiesterase, nuclease, esterase, protease, dehydrogenase, oxidase, and sulfatase) were performed in 96-well microplates at 70 °C as previously described (5Yang Z. Savchenko A. Yakunin A. Zhang R. Edwards A. Arrowsmith C. Tong L. J. Biol. Chem. 2003; 278: 8804-8808Google Scholar, 6Sanishvili R. Yakunin A.F. Laskowski R.A. Skarina T. Evdokimova E. Doherty-Kirby A. Lajoie G.A. Thornton J.M. Arrowsmith C.H. Savchenko A. Joachimiak A. Edwards A.M. J. Biol. Chem. 2003; 278: 26039-26045Google Scholar). Hydrolysis of single-stranded M13 DNA or double-stranded λ DNA or E. coli genomic DNA was assayed by analyzing reaction products by agarose gel electrophoresis. Hydrolysis of RNA (yeast RNA type III) was analyzed by following the change in absorbance at 260 nm after 30 min incubation with MJ0936. Hydrolysis of cyclic mononucleotides (2′,3′-cAMP, 2′,3′-cGMP, 3′,5′-cAMP, and 3′,5′-cGMP) was probed using Sigma 5′-nucleotidase and malachite green reagent to detect the released inorganic phosphate (7Vogel A. Schilling O. Niecke M. Bettmer J. Meyer-Klaucke W. J. Biol. Chem. 2002; 277: 29078-29085Google Scholar). Phosphatase activity toward diverse physiological phosphatase substrates (pyrophosphate, nucleotides, phosphorylated carbohydrates, and amino acids) was investigated by detection of released phosphate using the malachite green reagent (8Baykov A.A. Evtushenko O.A. Avaeva S.M. Anal. Biochem. 1988; 171: 266-270Google Scholar). Phospholipase C activity was analyzed using p-nitrophenylphosphoryl choline (pNPPC) 1The abbreviations used are: pNPPC, p-nitrophenylphosphoryl choline; pNPP, bis(p-nitrophenyl)phosphate; pNP, p-nitrophenyl phosphate; TMP-pNP, thymidine 5′-monophosphate p-nitrophenyl ester. (9Kurioka S. Matsuda M. Biochemistry. 1976; 75: 281-289Google Scholar). Phospholipase D activity against phosphatidylcholine was probed using choline oxidase (Sigma) and the peroxidase dianisidine-coupled assay as for oxidase testing (10Kelly R.L. Reddy C.A. Methods Enzymol. 1988; 161: 307-316Google Scholar). For phosphodiesterase kinetic studies, all phosphodiesterase assays were performed in a buffer containing 50 mm diethanolamine, pH 9.8, 40 mm NaCl, and MJ0936 (0.02 μg/ml) with different concentrations of substrates (bis-pNPP, pNP-TMP, and pNPPC) and metals (Ni2+ and Mn2+). The specific activity was determined as micromoles of product per min per mg of protein. The Lineweaver-Burk treatment of experimental data gave a linear plot of 1/V versus 1/[substrate]. According to the rearranged Michaelis-Menten equation, 1/V = 1/Vmax + Km/Vmax·1/[substrate], the Km and Vmax were determined from the Lineweaver-Burk plot using the GraphPad Prism program (version 4.00 for Windows, GraphPad Software, San Diego, CA), and kcat was determined by kcat = Vmax/[enzyme]. The optimum pH was examined in the presence of 50 mm glycine buffer, 40 mm NaCl, 7 mm Ni2+, 0.7 mm bis-pNPP, 0.1 μg of MJ0936, at pH values ranging from 8.6 to 10.4. Crystallization—Crystallization conditions were screened with a sparse matrix sampling method (11Jancarik J. Kim S.H. J. Appl. Crystallogr. 1991; 24: 409-411Google Scholar) using the hanging drop vapor diffusion technique at room temperature with commercially prepared reagents (Hampton Research, Laguna Niguel, CA). Selenomethionyl crystals of protein in the absence of exogenous metal (native crystals) were grown by vapor diffusion at 20 °C in a solution containing 15 mg/ml protein, 0.7 m ammonium sulfate. Magnesium-soaked crystals were obtained by soaking the native crystal with 20 mm magnesium sulfate in the crystallization drops. Co-crystals with magnesium, nickel, or manganese were grown by vapor diffusion at 20 °C in a solution containing 15 mg/ml protein, 0.7 m ammonium sulfate, 20 mm magnesium sulfate, or 5 mm nickel sulfate or 5 mm manganese sulfate. All the crystals were flash-frozen in a solution containing 0.7 m ammonium sulfate, 30% ethylene glycol and mounted on loops at 100 K prior to data collection. Structure Solution and Refinement—For native (unsoaked; selenium-Met) crystals, selenium edge (0.9790 Å) single anomalous dispersion data set was collected to 3.0 Å at the Advance Light Source beam line 5.0.2 using the ADSC Quantum 210 CCD detector placed 250 mm from the sample. High resolution data set was also collected to 2.4 Å at the Advance Light Source beam line 5.0.1 using the ADSC Quantum 210 CCD detector at a wavelength of 1.0 Å. The data were processed using programs HKL2000 and SCALEPACK (12Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Google Scholar). X-ray data statistics are shown in Table I. The 6 selenium sites were located using CNS (13Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Google Scholar). Phases were subject to 8 cycles of refinement and solvent flattening (14Wang B.-C. Methods Enzymol. 1985; 115: 90-112Google Scholar) followed by phase extension to 2.4 Å. The figure-of-merit after phase extension achieved 0.92 overall and 0.91 in the 2.4-Å resolution The was performed using M. Acta Crystallogr. Sect. A. 1991; Scholar). The was in the 2.4-Å experimental for a A containing residues was from of the using of phase combination and The was then to 2.4-Å resolution using CNS (13Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Google Scholar). The were used the of the data were for The refinement statistics are shown in Table have been at the Protein with in are to the resolution is Å for the native crystal and Å for the nickel and manganese and Å for the magnesium = in are to the resolution is Å for the native crystal and Å for the nickel and manganese and Å for the magnesium = in a new Table = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = fraction of of in a new For crystals, with magnesium, and with single wavelength data at wavelength were collected at the Advance Light Source beam line 5.0.1 using the ADSC Quantum 210 CCD detector placed mm from the sample. For with nickel, single wavelength data at the nickel edge Å) were collected at Advance Light Source beam line 5.0.2 using the ADSC Quantum 210 CCD detector placed mm from the sample. The data were processed using programs HKL2000 and SCALEPACK (12Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Google Scholar). X-ray data statistics are shown in Table I. The were from the structure of native MJ0936 crystal by the molecular method using J. Acta Crystallogr. Sect. A. Scholar). The were then to resolution using CNS (13Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Google Scholar). The were used the of the data were for The refinement statistics are shown in Table have been at the Protein with and for manganese and nickel The programs P. J. Appl. Crystallogr. 1991; 24: Scholar) and A. B. 1991; Scholar) were used for of the 50 sequence homologs of this protein have sequence ranging from 100 to homologs are and shown in This sequence that are in this protein (i) residues 7 to a (ii) residues to a (iii) residues to a residues to a residues to a and residues to a residues are conserved in the 10 shown in but were also found to be conserved in the homologs in this protein family. are these that most of these sequence may be involved in metal binding. Enzymatic of MJ0936 MJ0936 protein was probed for enzymatic activity in the presence of 5 mm using general enzymatic assays activity toward phosphatase, protease, esterase, dehydrogenase, or oxidase indicator substrates was but the protein but phosphodiesterase activity with in the presence of This substrate is into and no activity toward was found in phosphatase screens that MJ0936 is a phosphodiesterase and on Significant activity was also found with and MJ0936 phosphodiesterase activity with cAMP or or activity with various substrates double-stranded λ DNA or E. coli yeast activity toward MJ0936 activity at pH and had an absolute requirement for a divalent metal with Ni2+ and Mn2+ being most a of bis-pNPP, Ni2+ phosphodiesterase activity = = at an the other Mn2+ had affinity = 0.5 = 0.1 and its activity to at and concentrations of and phosphodiesterase activity was also with 0.5 mm or 20 mm To our MJ0936 is the phosphodiesterase. also the of the of the reaction on the substrate for bis-pNPP, TMP-pNP, and in the presence of 1 mm Ni2+ or 20 Mn2+ pNPPC, MJ0936 Michaelis-Menten with both but with or a was in the presence of Ni2+ the for both substrates show and for no was with substrate concentrations to 20 mm Ni2+, MJ0936 had the affinity to = but the kcat and the catalytic for was than for the other substrates kinetic for MJ0936 determined with Ni2+ or mm × mm × mm × × with 1 mm Ni2+ mm × with 1 mm Ni2+ (50 mm × with 1 mm Ni2+ in a new mass revealed the presence of phosphate in purified MJ0936, we the of phosphate on the of in the presence of 1 mm The is by phosphate with being at 30 mm in and the was and in a in Km for all residues of MJ0936, most of are well in the The been at 2.4-Å resolution to a of The from are Å for Å for and for The in for the is to be Å by a plot Acta Crystallogr. Scholar). The plot produced with R.A. Thornton J.M. J. Appl. Scholar) that of all residues in the are two in an by are to The of the MJ0936 is shown in The of × 40 × 30 MJ0936 is a protein. The of the protein is a cluster and with three on and on the other A and This protein is on the all crystal are two per and the of from crystal to the of the a large of or the from these are of Thus, we the of the is A L. C. 1996; Scholar) search using the crystal structure found the following homologs in the Protein J. Z. G. H. Nucleic Acids Res. 2000; Scholar) with than (i) A. L. Scholar) (ii) and phosphatase D. S. J. Scholar, T. P. H. B. Scholar) and (iii) and λ protein phosphatase A. Scholar, Biochemistry. 2000; Scholar) and and E. coli 5′-nucleotidase T. Biol. Scholar) All of these structural homologs have with MJ0936 at the sequence and they were using a BLAST W. W. J. Biol. Scholar) search against the sequence data the sequence they all have and molecular functions that are nuclease, phosphatase, or on the structural with structure homologs of MJ0936, we have determined that the active site is formed by a cluster of and All of these are conserved at the sequence and sequence involved in metal and the and refinement a putative was found in the active The be in the experimental single anomalous and To the bound in the active site of the purified protein, the mass method was The metal found was and no magnesium, nickel, or manganese was a of was detection The of the over MJ0936 is data is of resolution to a as to the of the bound at the active site of the native are two for the found in the active site to the of two and at the (i) an unknown metal (ii) phosphate in mass or sulfate in the crystallization the crystal structure suggested a biochemical all of divalent metal we crystals of the protein with divalent ions. Magnesium-soaked and have obtained crystals in 10 mm magnesium sulfate as well as of native protein in the presence of 20 mm magnesium sulfate. were collected and were determined for these crystals and The crystal of the protein in both the crystals and magnesium are to the native crystal structure produced in the absence of exogenous the of magnesium in the crystallization change the structure of the native protein. and and manganese were obtained by of MJ0936 and 5 mm nickel sulfate or manganese sulfate For the nickel single anomalous data were collected and metal sites were identified using anomalous sites in the manganese were identified using a and manganese well and have to The most the native crystal well as crystals and magnesium and the nickel or manganese are: (i) cell and crystal are (ii) per was in the native a cluster or was found in (iii) the overall structural native and crystals, are structural as shown in The structural are located in two (i) a and to and (ii) a and to The in the native crystal is shown in and in are shown in and for the active the most native and crystals is found at residues and the native these two residues are from the active site and an is found to to crystals, a change in the of these two residues is as a of a change in the and to the both and are in toward the active site and with a binuclear metal cluster to Structural 6 the structural with its structural as an to the and is a P. for the DNA A. L. Scholar). the overall sequence MJ0936 and is the active site residues are well conserved in both proteins as shown in The conserved sequence in MJ0936 are also found in sequence MJ0936 and its structure The conserved sequence found in MJ0936 also are shown the residues MJ0936 and are in residues in overall structure of the manganese of MJ0936 and MJ0936 is to domain of ions in both are as A the manganese ions in MJ0936 is as a A bound found in is also shown in the active site of MJ0936 as and as and are as and as and is of two A. L. Scholar) The fold and overall structure of domain are to of MJ0936, both of belong to the protein family. The active site in domain of is also to the active site in the residues at the active sites in the two are The in MJ0936 and are and The residues in the in MJ0936 and in are involved in metal in the active conserved active site residues have and This the that MJ0936 and a is to a in DNA substrate A. L. Scholar). nickel or manganese containing crystals, an was found two nickel or manganese metal ions in the and The and of this to the two metal or is to the of the phosphate in to two manganese in the crystal structure of the is for a we as a are three of on cyclic nucleotides, and our studies, MJ0936 no catalytic activity toward cyclic or Thus, the molecular function of the protein is most a with a novel phosphodiesterase activity. This is with the of its active site being to large we the in to the metal requirement for the the metal ions are most a of the the structure of suggests that they may have that of the product of the reaction by to the phosphate of the The of these two may be for the in metal shown in The native protein is and we as a phosphate bound in the active or protein is active and a cluster in the active active and may be by two and in and of the active as shown in the phosphate the and the cluster the active the of the Advance Light Source and for data collection. are also to and of for mass of Analysis of for Yokota and for and for studies and cell and for

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