The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

Abstract

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7–7.0. The enzyme had a Km of 0.53 mm for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond. Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7–7.0. The enzyme had a Km of 0.53 mm for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond. Organophosphonates are a group of biogenic compounds characterized by the presence of a stable covalent carbon to phosphorus (C–P) 1The abbreviations used are: C–P, covalent carbon to phosphorus bond; PEP mutase, phosphoenolpyruvate phosphomutase; PPH, phosphonopyruvate hydrolase; CAPS, 3-(cyclohexylamino)propanesulfonic acid. 1The abbreviations used are: C–P, covalent carbon to phosphorus bond; PEP mutase, phosphoenolpyruvate phosphomutase; PPH, phosphonopyruvate hydrolase; CAPS, 3-(cyclohexylamino)propanesulfonic acid. bond. Although they are believed to have originated before the earth's atmosphere became extensively oxygenated, C–P compounds have been isolated from a wide variety of extant life forms, and a significant percentage of biogenic phosphorus is believed to occur in the C–P linkage. Moreover a growing number of synthetic organophosphonates have found applications in industrial, agricultural, and domestic products and are ultimately disposed of to soils or natural waters. An understanding of C–P bond metabolism is thus a prerequisite to a fuller understanding of global biogeochemical phosphorus cycling. Four distinct bacterial enzymes are known to carry out the C–P bond cleavage reaction that is central to organophosphonate mineralization. The C–P lyase complex(es) has a broad substrate specificity (1Wanner B.L. Biodegradation. 1994; 5: 175-184Google Scholar) and can act upon unsubstituted alkyl and aryl organophosphonates (general formula R–PO3H2). The two C–P hydrolases that act on phosphonoacetaldehyde (OHC–CH2–PO3H2) (2La Nauze J.M. Rosenberg H. Shaw D.C. Biochim. Biophys. Acta. 1970; 212: 332-350Google Scholar) and phosphonoacetate (HOOC–CH2–PO3H2) (3McGrath J.W. Wisdom G.B. McMullan G. Larkin M.J. Quinn J.P. Eur. J. Biochem. 1995; 234: 225-230Google Scholar) are essentially specific to their respective substrates. A fourth C–P bond-metabolizing enzyme, phosphoenolpyruvate phosphomutase (PEP mutase), catalyzes the intramolecular rearrangement and interconversion of 3-phosphonopyruvate (COOH–CO–CH2–PO3H2) and phosphoenolpyruvate (COOH–C(=CH2)–O–PO3H2) (4Seidel H.M. Freeman S. Seto H. Knowles J.R. Nature. 1988; 335: 457-458Google Scholar). Phosphonopyruvate formation by this route is a key step in the biosynthesis of all known natural products that contain the C–P bond (5Ternan N.G. McGrath J.W. McMullan G. Quinn J.P. World J. Microbiol. Biotechnol. 1998; 14: 635-647Google Scholar). One of the most widely distributed of such biogenic C–P compounds is 2-amino-3-phosphonopropionic acid, commonly called phosphonoalanine (HOOC–CH(NH2)–CH2–PO3H2); it is formed through the transamination of phosphonopyruvate by many lower organisms, such as the sea anemone Zoanthus sociatus (6Kitteridge J.S. Hughes R.R. Biochemistry. 1964; 3: 991-995Google Scholar) and the protozoan Tetrahymena pyriformis (7Horigane A. Horiguchi M. Matsumoto T. Biochim. Biophys. Acta. 1980; 618: 383-388Google Scholar). Our earlier studies on its biodegradation showed that 47 of 100 randomly chosen environmental bacterial isolates had the ability to utilize phosphonoalanine as sole phosphorus source for growth (8McGrath J.W. Ternan N.G. Quinn J.P. Lett. Appl. Microbiol. 1997; 24: 69-73Google Scholar). Subsequently Ternan et al. (9Ternan N.G. Hamilton J.T.G. Quinn J.P. Arch. Microbiol. 2000; 173: 35-41Google Scholar) reported the isolation of an environmental Burkholderia cepacia strain capable of growth on phosphonoalanine as sole source of carbon, nitrogen, and phosphorus. Cell extracts of the isolate contained a previously unknown Pi-independent activity designated phosphonopyruvate hydrolase (PPH) that catalyzed the hydrolytic cleavage of the C–P bond of phosphonopyruvate to yield pyruvate and Pi (Fig. 1). We now describe the purification and properties of PPH from a soil isolate of a Variovorax sp. and the sequence analysis of the structural gene that encodes it. Intriguingly, the enzyme shares a common ancestry with PEP mutase, which is responsible for phosphonopyruvate biosynthesis (see above); the interaction of the two activities is thus likely to play an important regulatory role in global phosphorus cycling. Microorganism and Culture Conditions—Samples from soils, natural water bodies, and waste treatment installations in Northern Ireland were incubated in mineral salts medium (8McGrath J.W. Ternan N.G. Quinn J.P. Lett. Appl. Microbiol. 1997; 24: 69-73Google Scholar) with d,l-phosphonoalanine (5 mm) as sole carbon, nitrogen, and phosphorus source for 5–7 days. Cultures were plated on the same medium solidified with agar, single colonies were isolated, and those that grew most rapidly were chosen for further study. Cells of Variovorax sp. Pal2 were routinely grown in batch culture at 30 °C on an orbital shaker at 100 rpm in mineral salts medium (8McGrath J.W. Ternan N.G. Quinn J.P. Lett. Appl. Microbiol. 1997; 24: 69-73Google Scholar) supplemented with 3 g pyruvate/liter and 2 g gluconate/liter as carbon sources, 5 mm NH4Cl as nitrogen source, and 0.5 mm phosphonoalanine as phosphorus source. Cells were harvested in mid-log phase by centrifugation, washed twice in 20 mm HEPES-KOH buffer, pH 7.0, and stored at –20 °C. Synthesis of 3-Phosphonopyruvate—The trilithium salt of phosphonopyruvate was prepared according to the method of Sparkes et al. (10Sparkes M.J. Rogers K.L. Dixon H.B.F. Eur. J. Biochem. 1990; 194: 373-376Google Scholar) by Chemical Synthesis Services, Belfast, Northern Ireland via chemical transamination of phosphonoalanine (Sigma) in the presence of Cu2+ and pyridine catalysts with glyoxylate as the amino group acceptor. The synthesis yielded a pale yellow solid, which was characterized by 31P and 1H NMR spectroscopy; this showed the material to be 3-phosphonopyruvate with a purity of 95% by 1H NMR and high pressure liquid chromatography. Enzyme Assay—PPH activity was assayed at 37 °C by measuring the amount of phosphate liberated from phosphonopyruvate. The assay mixture (1 ml) contained 100 μmol HEPES-KOH buffer, pH 7.0, 5 μmol phosphonopyruvate, and 5 μmol CoCl2. The reaction was initiated by the addition of 25 μl of enzyme solution (0.1–0.3 μg of protein/ml) and terminated after 10 min by the addition of 200 μl of 3 m trichloroacetic acid. The mixture was centrifuged, and the phosphate released was measured by the method of Fiske and Subbarrow (11Fiske C.H. Subbarrow Y. J. Biol. Chem. 1925; 66: 375-400Google Scholar). Activities were expressed as units of 1.0 μmol of phosphate released per min (mean of three replicates). When required, pyruvate release was measured using a lactate dehydrogenase-based pyruvate test kit (Sigma). Protein Assay—Protein concentrations were determined by the BCA (bicinchoninic acid) method with bovine albumin as standard (Pierce). Purification of Phosphonopyruvate Hydrolase—During enzyme purification all column chromatography steps, each lasting for no more than 2.5 h, were performed at room temperature. Collected fractions were removed to ice. For preparation of crude extract, the washed cells (∼15 g wet weight from 5 liters of the culture) were suspended in 65 ml of 20 mm HEPES-KOH buffer, pH 7.0, and disrupted by sonication on ice for 6 min (30-s sonication followed by 2-min cooling) at 16 kHz. The cellular debris was removed by centrifugation (25,000 × g for 30 min at 4 °C), and the supernatant was immediately used in the next step. For ammonium sulfate fractionation, the cell-free supernatant was fractionated in two steps by addition of solid ammonium sulfate at 20 °C and stirring for 1 h. The precipitate obtained between 2.1 and 3.1 m was collected by centrifugation (10,000 × g for 30 min at 4 °C) and dissolved in 20 mm HEPES-KOH buffer, pH 7.0. For hydrophobic interaction chromatography, the protein fraction obtained by salt precipitation was applied to a phenyl-Sepharose HP column (5.0 × 4.0 cm) (Amersham Biosciences) equilibrated with 50 mm HEPES-KOH buffer, pH 7.0, containing 1 m (NH4)2SO4. The column was washed with 3 bed volumes of 0.5 m (NH4)2SO4 in 50 mm HEPES-KOH buffer, pH 7.0. The proteins were eluted at 8 ml/min first with 0.1 m (NH4)2SO4 in 50 mm HEPES-KOH buffer, pH 7.0 (200 ml), and then the enzyme with the buffer alone (160 ml). Fractions containing active enzyme were pooled, concentrated, and desalted with 20 mm HEPES-KOH buffer, pH 7.0, by ultrafiltration through a Vivaspin 15 concentrator (10,000 molecular weight cut-off) (Sartorius). For anion-exchange chromatography (column 1), the enzyme solution was applied to a Q-Sepharose HP anion-exchange column (1.6 × 4.8 cm) (Amersham Biosciences) equilibrated with 20 mm HEPES-KOH buffer, pH 7.0. Proteins were eluted at 2.5 ml/min, applying an initial isocratic step in the equilibration buffer (27 ml) followed by a linear gradient of 0–0.2 m NaCl in the same buffer (80 ml). Fractions containing the enzyme were pooled and dialyzed against 25 mm HEPES-KOH buffer, pH 7.0, containing 0.5 m NaCl. For immobilized metal ion affinity chromatography, the enzyme solution (1.1 ml) was applied to a chelating-Sepharose column (1.0 × 7.5 cm) (Amersham Biosciences) charged with Cu2+ and equilibrated with 25 mm HEPES-KOH buffer, pH 7.0, containing 0.5 m NaCl. The column was washed with the same buffer (20 ml), and the bound proteins were eluted at 2 ml/min with a linear gradient of 0–0.5 m NH4Cl in 25 mm HEPES-KOH buffer, pH 7.0, containing 0.5 m NaCl. The active fractions were pooled, concentrated, and desalted with 20 mm HEPES-KOH buffer, pH 7.8, by ultrafiltration as before. For anion-exchange chromatography (column 2), the sample (1.1 ml) was applied to a Q-Sepharose HP column (1.0 × 4.5 cm) equilibrated with 20 mm HEPES-KOH buffer, pH 7.8. The column was washed with the same buffer (10 ml), and the bound proteins were removed with a linear gradient of 0–0.2 m NaCl in the same buffer at a flow rate of 1 ml/min. PPH was eluted in a single major peak at about 0.19 m NaCl. The active fractions were pooled, dialyzed, and concentrated by ultra filtration as before and stored in aliquots with 25% glycerol at –20 °C. Gel Electrophoresis—SDS-PAGE used precast 8–16% Tris/glycine or NuPAGE BisTris gels in an X cell 11 mini-cell with Mark 12 molecular mass standards (Invitrogen). Gels were stained with EZBlue gel staining reagent (Sigma) or the kit (Invitrogen). used precast 8–16% Tris/glycine gels (Invitrogen). Enzyme activity was by of the gel for 15 min at 37 °C in the phosphonopyruvate hydrolase assay mixture and then the phosphate released with the Fiske and Subbarrow reagent (see above); the activity was by a Protein was using the EZBlue reagent (Sigma). was performed using an column equilibrated with 20 mm phosphate buffer, pH containing m at a flow rate of 1 ml/min. Gel gels at pH were used with standards Protein 50 μg of enzyme was dissolved in acid and water and a of was at room for the sample was and dissolved in 10 μl of sample enzyme and its were in NuPAGE BisTris gels and by the method using 10 mm buffer, pH containing Protein were by EZBlue (Sigma) staining and was performed by the The was out with a Enzyme of on the of PPH was determined by of the reaction mixture substrate at from 30 to °C for 10 min to the of pH optimum was determined using the standard assay with of 100 mm as pH pH pH and pH of activity was assayed at the from to °C. of of metal on purified enzyme that had been dialyzed was by the addition of (1 mm) for 1 at °C to the reaction mixture and then metal (5 mm for 5 min at 37 °C to measuring The activity in the dialyzed preparation was and after treatment this to 30 all activities were with the of and Km of the purified enzyme were for phosphonopyruvate by using concentrations from to Activity was measured at 37 °C as and of the isolation and were out J. A Scholar). and with were performed as by the (Amersham and was used for of the and containing from the gene and were used as for the kit were obtained using an The nucleotide of were analysis of the was performed using the The of was performed by using 1994; Scholar) with at the of the amino acid sequence of the purified enzyme, a was A was using the sequence of the A of the gene was by using Variovorax sp. Pal2 and and The reaction was out in a of 25 μl with concentrations of at 200 and the and at The was initial at °C for 1 min and then 30 of °C for °C for and °C for Subsequently the gene was used as a for the were with and of Variovorax sp. Pal2 were to (Amersham Biosciences) by and the were then according to the of the were using the and (Amersham was out at and was performed according to the on the of of and of Variovorax sp. Pal2 were in and containing with the gene were by with the same 16 16 gene from isolate Pal2 was by with the by et al. J. 1995; Scholar). of the 16 gene and its analysis were out as previously K.L. Larkin M.J. Appl. Microbiol. in Scholar). of Variovorax sp. of culture on d,l-phosphonoalanine was out using from soils, natural water bodies, and waste treatment capable of the as sole carbon, nitrogen, and phosphorus source were obtained from all the isolates were found to utilize the of A number of isolates were obtained that were capable of growing on phosphonopyruvate, the transamination of as the sole carbon and phosphorus source. Enzyme out on crude extracts of the that each strain contained PPH which was inducible in the presence of phosphonoalanine or phosphonopyruvate or of the enzyme was in isolate by the presence of The bacterial designated that grew most on phosphonoalanine was chosen for further it was by 16 sequence as a Variovorax sp. Purification of studies on cell extracts from Variovorax sp. Pal2 a in activity on at room for h. was to to and all chromatography fractions were on ice. A was to this a of salt precipitation and chromatography using hydrophobic immobilized metal and The PPH was purified a and had a specific activity of of Enzyme prepared in this as a single protein of kDa on gels (Fig. When Tris/glycine gels were used the PPH activity was found to with the single on staining (Fig. of phosphonopyruvate after by pH after by after by pH after by after by in a The of enzyme had a molecular mass of kDa determined by mass and 63 kDa by chromatography that PPH is a The of the purified protein was The of the and two by and were determined of the amino and acid was to be the amino acid the cleavage used for the of for are was to be the amino acid the cleavage used for the of for are in a The of of on the enzyme showed that activity was after an of 10 min to 50 °C. The of enzyme was by of 25% and after 6 of at –20 °C the in activity was The of and on The of pH on the reaction was the pH from to and an optimum of about 7.0 was The of was and 37 °C was for the hydrolases for compounds were to be (2La Nauze J.M. Rosenberg H. Shaw D.C. Biochim. Biophys. Acta. 1970; 212: 332-350Google J.W. Wisdom G.B. McMullan G. Larkin M.J. Quinn J.P. Eur. J. Biochem. 1995; 234: 225-230Google the of treatment and metal ion addition to the dialyzed enzyme were to activity by about to and was found to PPH and had no whereas and Cu2+ the activity by and the enzyme is on and this ion was in the assay of metal on phosphonopyruvate hydrolase (5.0 in a and of the catalyzed reaction was by the release of pyruvate and phosphate from phosphonopyruvate with the purified The ability of the enzyme to the intramolecular rearrangement of phosphonopyruvate to phosphoenolpyruvate was using the of et al. H. A. T. Seto H. J. this a rate of phosphoenolpyruvate formation lower than that of pyruvate and as a substrate for phosphonopyruvate phosphatase and acid) were as PPH were 20 compounds activity was with phosphonopyruvate. The of the compounds on the purified enzyme were was found that 3-phosphonopropionic acid hydroxymethylphosphonic acid and phosphonoformic acid significant 5 in the of the phosphonoacetate hydrolase (3McGrath J.W. Wisdom G.B. McMullan G. Larkin M.J. Quinn J.P. Eur. J. Biochem. 1995; 234: 225-230Google Pi the The enzyme had an Km of 0.53 mm for its sole substrate, phosphonopyruvate, and a of units per of of addition of compounds on phosphonopyruvate hydrolase (5 activity was of Activity are the of activity was of Activity are the of in a of the amino acid of PPH and its by were used to for analysis of the PPH with and yielded a of which was and was used as a for of and containing the gene were were contained a and a The nucleotide sequence of the gene was gene encodes a PPH protein of amino with an molecular mass of is in with the molecular mass obtained for the purified PPH by mass The of the PPH protein and the two of its to amino acid of The of the gene was by with the PPH amino acid further that the gene encodes phosphonopyruvate hydrolase, cell extracts from of containing and were prepared and for PPH activities of and of protein were found in cells that contained and PPH activity was found in the of and with the S. A. 1988; Scholar) and showed that the PPH protein has with a number of of the PEP The were with PEP from T. pyriformis H. T. Seto H. Biochim. Biophys. Acta. 2000; Scholar) amino acid sequence and Y. Biochemistry. Y. Scholar) A lower level of identity was found with the bacterial PEP from were found between PPH and lyase from and lyase of (Fig. of the PPH sequence with proteins to the PEP superfamily is in The in of the superfamily and the active of M. PEP are as by and Y. Biochemistry. Scholar). Although are of between PPH and the PEP superfamily is a of amino acid between PPH and PEP in (Fig. and is that most of the amino acid that the active of M. PEP Y. Biochemistry. Scholar) are in PPH from Variovorax sp. Pal2. Phosphonopyruvate is through the rearrangement of the high phosphate by PEP mutase, the major C–P bond-forming enzyme to Thus the is central to the formation of all natural phosphonopyruvate be to phosphonoalanine H. T. Seto H. J. 1997; the initial step in all of the major of natural products is believed to be its to phosphonoacetaldehyde by phosphonopyruvate H. T. Seto H. J. 1997; Scholar). the of the reaction out by PEP phosphoenolpyruvate this to the reaction in the of C–P bond formation H.M. Knowles J.R. Biochemistry. 1994; Scholar). with this is the of the expression of the PEP and phosphonopyruvate in the strain S. J. S. Microbiol. Lett. 1998; Scholar) and the H. T. Seto H. Biochim. Biophys. Acta. 2000; Scholar). The of PPH, a novel C–P bond cleavage enzyme of apparently substrate specificity purification and properties now the of an for phosphonopyruvate; the activity of this enzyme has now been by in cell extracts of a of environmental capable of the of phosphonoalanine as sole carbon, nitrogen, and phosphorus source or of phosphonopyruvate as sole carbon and phosphorus source PPH is the bacterial capable of C–P bond cleavage to be to its for a metal ion it has to many enzymes the C–P hydrolases specific to phosphonoacetate (3McGrath J.W. Wisdom G.B. McMullan G. Larkin M.J. Quinn J.P. Eur. J. Biochem. 1995; 234: 225-230Google Scholar) and phosphonoacetaldehyde Biol. 2000; the is zinc-dependent whereas in the is a in a that an active and formation of a intermediate Biol. 2000; Scholar). PPH is to the two hydrolases in its the subunit are the phosphonoacetate hydrolase is 30 kDa whereas the phosphonoacetaldehyde hydrolase is kDa. of the of the three enzymes that they have Thus whereas phosphonoacetaldehyde hydrolase belongs to the dehalogenase that M.J. B.L. Biochemistry. 1998; and phosphonoacetate hydrolase is a member of the alkaline phosphatase superfamily of zinc-dependent hydrolases A. Protein 1998; PPH (Fig. to the of a enzyme superfamily Y. Biochemistry. Scholar) that lyase and PEP in of their common substrate, PPH showed of amino acid sequence identity with the PEP in the active in the PEP of the M. A. J. J. Biol. Chem. 1998; Scholar) are in PPH, as they are in of the Y. Biochemistry. Scholar). amino the and of the M. enzyme that have been in group and Y. Biochemistry. Scholar). PPH a ability to phosphonopyruvate to PEP it is to release Pi from the The that the of was found between the PPH of Variovorax sp. Pal2 and the PEP from the lower M. and T. pyriformis to the ancient of studies the in which their two active have to their in the interaction of the two activities they occur in the same whereas the of each with phosphonopyruvate We the of G. J. with We are to J. for the chromatography and for out the mass We for and in this study.