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Originally published In Press as doi:10.1074/jbc.M413759200 on December 16, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5274-5280, February 18, 2005
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A Nudix Enzyme Removes Pyrophosphate from Dihydroneopterin Triphosphate in the Folate Synthesis Pathway of Bacteria and Plants*{boxs}

Sebastian M. J. Klaus{ddagger}§, Arno Wegkamp§, Wilbert Sybesma¶, Jeroen Hugenholtz¶, Jesse F. Gregory, III||, and Andrew D. Hanson{ddagger}**

From the {ddagger}Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, the Department of Flavor, Nutrition and Natural Ingredients, Wageningen Centre for Food Sciences, NIZO Food Research, Kernhemseweg 2, P. O. Box 20, 6710, BA Ede, The Netherlands, and the ||Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida 32611

Received for publication, December 7, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase. Because many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized. The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a Km value of 2 µM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (~1 mM) of Mg2+, and was active as a monomer. Essentially no reaction occurred without enzyme at 1 mM Mg2+. Inactivation of ylgG in L. lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis. We therefore propose that ylgG be redesignated as folQ. The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E. coli and shown to have Mg2+-dependent DHNTP pyrophosphohydrolase activity. This protein (AtNUDT1) was reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn2+ (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482–50486). However, we found that this activity is negligible at physiological levels of Mn2+ and that, with 1 mM Mg2+, AtNUDT1 prefers DHNTP and (deoxy) nucleoside triphosphates.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Enzymes and genes for all except one of the specific steps of the folate biosynthesis pathway have been identified in bacteria (1) and plants (2–7). The exception is the second step of the pterin branch of the pathway, in which pyrophosphate is cleaved from dihydroneopterin triphosphate (DHNTP),1 yielding dihydroneopterin monophosphate (DHNP) (Fig. 1A). Furthermore, there has been controversy over whether this step is enzymatic (8–10).



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  		FIG. 1.
Schemes of the reaction that removes pyrophosphate from DHNTP (A) and of the multicistronic folate biosynthesis operon of L. lactis (B). The folKE gene encodes a bifunctional GTP cyclohydrolase I/hydroxymethyldihydropterin pyrophosphokinase; folP encodes dihydropteroate synthase; folC encodes dihydrofolate synthase, and ylgG encodes a Nudix protein.

 
In favor of an enzymatic mechanism, Suzuki and Brown (9) partially purified a small (17 kDa) Escherichia coli protein able to catalyze the release of pyrophosphate from DHNTP but not nucleoside triphosphates. The enzyme was accordingly named DHNTP pyrophosphohydrolase. The Km for DHNTP was 11 µM, and activity was Mg2+-dependent. No evidence was presented showing this activity to be necessary for folate synthesis, however. More recent work in Bacillus subtilis has questioned the requirement for an enzyme, because chemical conversion of DHNTP to DHNP was observed at pH 8 and 37 °C in the presence of Mg2+ or Ca2+ (10). Unfortunately, the Mg2+ and Ca2+ concentrations studied were unphysiologically high (5–12 mM), with typical cytosolic levels of free Mg2+ and Ca2+ being ~1 mM and ~0.1 µM, respectively, in both bacteria and plants (11–14).

The mechanism of the DHNTP -> DHNP step is of considerable interest. First, if there is a specific DHNTP pyrophosphohydrolase, it could be a novel target for antibacterial drug discovery in the folate synthesis pathway (8). Second, modestly higher fluxes to folate can be engineered in bacteria (15) and plants (16, 17) by greatly increasing the production of DHNTP, but it is not yet clear whether this DHNTP is efficiently converted to DHNP. If it is not, engineering the overexpression of a DHNTP pyrophosphohydrolase could be used to correct the problem.

The annotated genome sequence of the lactic acid bacterium Lactococcus lactis (18) offers a clue to the existence of a specific DHNTP pyrophosphohydrolase, for it reveals a folate biosynthesis gene cluster in which known folate genes flank a gene (ylgG) encoding an unknown protein from the Nudix hydrolase family. Nudix (nucleoside diphosphate linked to x) hydrolases are typically small proteins (15–25 kDa), contain a characteristic motif (GX5EX7REUXEEXGU, where U is a hydrophobic residue), and many have nucleoside triphosphate pyrophosphohydrolase activity (19). Furthermore, ylgG was shown to be cotranscribed with three of the flanking folate genes in a multicistronic operon (15, 20) (Fig. 1B). Genomic evidence thus identifies YlgG as a candidate for the elusive DHNTP pyrophosphohydrolase.

In this study, we set out to find whether recombinant L. lactis YlgG has specific DHNTP pyrophosphohydrolase activity, and whether ylgG deletants are defective in pteridine and folate synthesis. When this proved to be the case, we sought homologs of YlgG among the Nudix proteins of Arabidopsis (21), and we showed that the closest homolog has DHNTP pyrophosphohydrolase activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—Pteridines were obtained from Schircks Laboratories (Jona, Switzerland). The Biomol GreenTM phosphate assay reagent was from Biomol (Plymouth Meeting, PA). Ni2+-nitriloacetic acid superflow resin was from Qiagen (Valencia, CA). PD-10 columns were from Amersham Biosciences. Oligonucleotides were from MWG Biotech (High Point, NC). Reduced nicotinamide mononucleotide (NMNH) was a gift of G. Magni (University of Ancona, Italy). Other chemicals were from Sigma or Fisher.

Inactivation of ylgG by Double Crossover Recombination—The bacterial strains and plasmids used are listed in Table I. E. coli was grown at 37 °C in TY medium (22), and L. lactis was grown at 30 °C in M17 medium (23) containing 0.5% glucose (GM17 medium) or in chemically defined medium (CDM), prepared as described (24) except that folic acid was omitted. E. coli was transformed by heat shock and L. lactis by electroporation (25). For E. coli, kanamycin and erythromycin were used at final concentrations of 50 and 200 µg/ml, respectively. PCRs were performed with Pfx polymerase (Invitrogen). The pre-integration vector for inactivation of ylgG was constructed by using pORI280 (26) and an amplified linear DNA fragment (2287 nucleotides) consisting of 450 bp of the 3'-end of folP, the entire ylgG gene, and the entire folC gene, by using primers folPNcoI-F and folCXbaI-R, which contained an NcoI site and an XbaI site, respectively (Table II). The amplicon was digested with NcoI and XbaI and cloned between the matching sites in pORI280, giving plasmid pORI280-folP-ylgG-folC. This plasmid was transformed to E. coli and harvested. The final integration vector, containing an in-frame deletion of almost the entire ylgG gene, was obtained by PCR amplification using pORI280-folP-ylgG-folC as a template and primers ylgGPstI-F and ylgGPstI-R (Table II). Both primers contained a terminal PstI site. The amplicon was digested with PstI and ligated, generating plasmid pORI280-folP-folC, which was transformed to E. coli and isolated for subsequent introduction into L. lactis strain NZ9000 (27). L. lactis transformants in which plasmids had integrated via single crossover were grown overnight in GM17 with 2 µg/ml erythromycin and plated on GM17 agar containing 2 µg/ml erythromycin and 80 µg/ml 5-bromo-4-chloro-3-indoyl-galactopyranoside (X-gal). The orientation of the single crossover event was tested by PCR amplifications directly on cell material from a blue colony using the primers pORIc-ntr-F (sequence present downstream of multiple cloning site of pORI280) and folP-F (sequence present in folP but not the integration vector) (Table II). Subsequently, cells that contained the plasmid integrated over the short folP-flanking region were grown in medium without antibiotics and re-inoculated in the same medium to a density of ~1–10 cells/ml. After growth for ~30–40 generations, dilutions of the culture were plated on agar containing X-gal. After 48 h white colonies were selected, and the presence or absence of ylgG was determined by PCR amplification from cell material by using primers folP-F and folCXbaI-R (Table II).


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  		TABLE I
Bacterial strains and plasmids used in ylgG inactivation

 


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  		TABLE II
PCR primers used in the inactivation of ylgG

Nucleotides in lowercase letters represent modifications with regard to the native gene. Restriction sites are underlined.

 
Gene Cloning and Protein Expression in E. coli—The ylgG gene was amplified from a pNZ8048 derivative (28) using KOD polymerase (Novagen, Madison, WI) and the primers 5'-CATGCACCATGGATGAGGATTTGATTTCT-3' (forward) and 5'-CCCGCTCGAGTATGTTCTTATAACGATA-3' (reverse), containing NcoI and XhoI sites, respectively. Using the NcoI site for cloning changed the second amino acid of YlgG from Asn to Asp. A full-length cDNA (Arabidopsis Biological Resource Center U50379 [GenBank] ) served as template to amplify the At1g68760 coding sequence, using the primers 5'-ACCCACATGTCGACAGGAGAAGCG-3' (forward) and 5'-CCCGCTCGAGGTCTCCACCACCATGAGT-3' (reverse), containing AflIII and XhoI sites, respectively. The ylgG and At1g68760 amplicons were digested with appropriate enzymes and cloned between the NcoI and XhoI sites of pET-28b (Novagen), which added a hexahistidine tag to the C terminus. The sequence-verified constructs were electroporated into E. coli BL21-CodonPlus®-RIL (DE3) cells (Stratagene, La Jolla, CA). For enzyme production, cells were grown at 37 °C in 1 liter of LB medium containing 100 µg/ml ampicillin and 20 µg/ml chloramphenicol until the A600 reached 0.5. Isopropyl-D-thiogalactopyranoside was then added (final concentration 1 mM) and incubation continued for 16 h at 25 °C.

Protein Isolation and Molecular Mass Determination—E. coli cells from a 1-liter culture were harvested by centrifugation, resuspended in 20 ml of 50 mM Tris-HCl, pH 8.0, containing 0.1 M KCl (buffer A), and broken in a Mini-BeadBeater (Biospec Products, Bartlesville, OK) using 0.1-mm zirconia/silica beads. Protein purification by Ni2+-affinity chromatography under native conditions followed the manufacturer's protocol (Qiagen). All steps were carried out at 0–4 °C. Purified proteins were desalted on a PD-10 column equilibrated in buffer A. For YlgG, a second purification step was carried out using a Waters 626 high pressure liquid chromatography system. Proteins were loaded on a MonoQ HR 5/5 column (Amersham Biosciences) equilibrated in buffer A containing 0.1 M NaCl. Proteins were eluted with a linear gradient of 0.1–0.5 M NaCl (0.2 ml/min), and fractions with DHNTP pyrophosphohydrolase activity were pooled. Purified proteins were stored at 4 °C for up to 6 months, which did not cause significant activity loss. Protein concentration was determined by Bradford's method (29) using bovine serum albumin as standard. The native molecular mass of YlgG was estimated using a calibrated Superdex 200 HR 10/30 column (Amersham Biosciences) equilibrated with 50 mM HEPES-KOH, pH 8.0, containing 150 mM NaCl, using a flow rate of 0.3 ml/min. The standards were carbonic anhydrase, bovine serum albumin, {beta}-amylase, apoferritin, and thyroglobin.

Preparation of DHNTP—Recombinant E. coli GTP cyclohydrolase I (30) was used to prepare DHNTP. The reaction was run in 1.5 ml of 50 mM HEPES-KOH containing 0.1 M KCl (buffer B) plus 1 mM GTP, 10 mM {beta}-mercaptoethanol, and 30 µg of purified GTP cyclohydrolase I for 2 h at 37 °C. DHNTP formation was followed by absorption at 330 nm until all GTP was converted; the molar extinction coefficient of DHNTP is 6300 M–1 cm–1 at 330 nm (31). The solution was then deproteinized using a Centricon YM-10 unit (Millipore, Billerica, MA). Aliquots containing 30–80 nmol of DHNTP were frozen in liquid N2 and stored at –80 °C. After thawing, aliquots were used at once and not re-frozen.

Enzyme Assays—Assays were routinely made in 50–200-µl reaction mixtures at 30 °C for 5–180 min in buffer B containing the specified concentrations of substrates and divalent metal ions (as chloride salts). The 0.1 M KCl in this buffer did not significantly affect enzymatic reactions. DHNTP pyrophosphohydrolase activity was assayed by fluorometric HPLC. Reactions were stopped by adding oxidizing solution (0.5% I2, 1% KI, w/v, in 1 M HCl, 10 µl per 100-µl reaction mix) and incubating for 1 h at 4 °C. Then 10 µl of 2% sodium ascorbate and 4.1 µl of 1 M Na2HPO4, 1 M NaOH were added to destroy excess I2 and adjust the pH to 6.0, and denatured protein was pelleted. Separation of neopterin and its phosphates was basically as described (4), using a Waters 2695 instrument (Waters, Milford, MA). Briefly, samples (20–50 µl) were injected on a C18 Synergi 4µ Fusion-RP 80 column (4 µm, 4.6 x 250 mm, Phenomenex, Torrance, CA) and eluted isocratically with 10 mM sodium phosphate buffer, pH 6.0, at 1 ml/min. Peaks were detected using a Waters 2475 Multi {lambda} Fluorescence Detector (excitation 350 nm, emission 450 nm) and quantified relative to neopterin monophosphate (Schircks). A neopterin diphosphate standard was prepared from DHNTP by acid phosphatase treatment and oxidation. Pyrophosphate was quantified using the Biomol GreenTM phosphate detection reagent after addition of 1–2 units of inorganic pyrophosphatase (Sigma) to enzymatic assays (32). To measure NADH hydrolysis, samples (20 µl) were injected on a C18 symmetry column (3.5 µm, 4.6 x 250 mm, Waters) and eluted in 50 mM sodium phosphate, pH 6.0, containing 8 mM tetrabutylammonium bisulfate using a linear gradient from 12 to 30% methanol. The NMNH peak was quantified fluorometrically (excitation 340 nm, emission 460 nm) relative to authentic NMNH. Hydrolysis of nucleoside triphosphates and their 2'-deoxy derivatives was assayed by pyrophosphate release as described above. Reactions were stopped by adding either excess EDTA (2–3 times the quantity of M2+ ions) or the Biomol GreenTM reagent. Hydrolysis of other substrates (Ap4A, Ap3A, NAD, ADP-ribose, UDP-glucose, and coenzyme A) was assayed by treating the reaction products with shrimp alkaline phosphatase (Roche Applied Science) as described (32) and determining phosphate with the Biomol GreenTM reagent. Kinetic data were analyzed by direct fitting to the Michaelis-Menten equation using nonlinear regression.

Folate and Pteridine Analysis—Total folate was quantified using the Lactobacillus casei microbiological assay (33) after enzymatic deconjugation, as described previously (15). Pteridines were extracted from L. lactis cells in buffer C (0.2 M sodium citrate, pH 4.5, containing 5 mM {beta}-mercaptoethanol) and analyzed by HPLC. Briefly, cells of a 50-ml overnight culture (30 °C) were harvested by centrifugation, resuspended in buffer C, and broken in a Mini-BeadBeater. Samples were centrifuged, and the supernatant was incubated for 10 min at 37 °C plus or minus 3 units of wheat germ acid phosphatase (Sigma). Acid hydrolysis conditions were used to avoid formation of cyclic DHNP, which is not attacked by phosphatase (4). Reactions were stopped by adding 15 µl of oxidizing solution per 100-µl sample and then incubated for 1 h at 4 °C. Then 15 µl of 2% sodium ascorbate and 6.1 µl of 1 M Na2HPO4, 1 M NaOH were added, and the samples were centrifuged and analyzed by HPLC as described above except that the flow rate was 1.5 ml/min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inactivation of ylgG—The ylgG gene was inactivated in L. lactis strain NZ9000{Delta}ylgG by double crossover recombination using modifications of a published method (26). The NZ9000{Delta}ylgG strain, which lacks 95% of the ylgG gene, had the same growth rate as the wild type on minimal (CDM) medium (Fig. 2A, inset). However, the mutant had intracellular folate levels 3-fold lower than the wild type and, unlike the wild type, secreted almost no folate to the medium (Fig. 2A). The complementation of ylgG into the NZ9000{Delta}ylgG strain restored folate level to that of the wild type (not shown). These data indicate that YlgG mediates a step in folate biosynthesis. HPLC analysis showed this step to be dephosphorylation of DHNTP, for mutant cells accumulated a pteridine with the retention time of DHNTP (Fig. 2B) that gave dihydroneopterin (DHN) upon phosphatase treatment (Fig. 2B and Table III). (Pteridines were oxidized before HPLC to convert them to their fluorescent aromatic forms, so that DHN was analyzed as neopterin and DHNTP as neopterin triphosphate.) Two other features of Fig. 2B are noteworthy. First, the mutant cells accumulated dihydroneopterin di- and monophosphates as well as DHNTP, showing that sequential removal of single phosphate residues was occurring. Second, wild type cells contained 10-fold more DHNTP than dihydroneopterin, showing that the dephosphorylation process is far from equilibrium in vivo.



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  		FIG. 2.
Effect of ylgG inactivation on L. lactis folate content, growth, and pteridine profile. A, intracellular levels (intra) and extracellular secretion (extra) of folate in wild type strain NZ9000 and mutant strain NZ9000{Delta}ylgG ({Delta}ylgG) grown in 10 ml of CDM for 18 h to an A600 value of 2.1 ± 0.1. Total folate was assayed microbiologically. Data are means of 6–10 determinations and S.E. Inset shows the doubling time of each strain and S.E. B, HPLC analysis of oxidized dihydroneopterin and its phosphates from strains NZ9000 and NZ9000{Delta}ylgG, without (solid lines) or with (broken lines) phosphatase treatment. N, neopterin; NP3, neopterin triphosphate; NP1, neopterin diphosphate; NP2, neopterin phosphate. The phosphatase-insensitive peak at 3.7 min that is higher in the mutant strain is an unidentified pteridine.

 


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  		TABLE III
Effect of ylgG inactivation on intracellular pteridine levels in L. lactis

Cultures (50 ml) were grown in CDM medium to an A600 value of 3. One-half of the pteridine extract of the cell pellet was treated with acid phosphatase (see "Experimental Procedures"); the other half was not. Pteridines were then oxidized to their aromatic forms and quantified by fluorometric HPLC. Values are means of three determinations ± S.E. and are expressed as intracellular concentrations. The internal cell volume of a 50-ml culture at A600 = 3 was estimated as 93 µl (taking an A600 value of 1 as equivalent to 0.2 mg of protein ml-1 and L. lactis internal cell volume as 3.1 µl mg-1 protein).

 
Characterization of Recombinant YlgG—The ylgG open reading frame (Fig. 3A) was expressed in E. coli with a C-terminal hexahistidine tag. The expression level was insufficient to detect the recombinant YlgG in the total soluble protein fraction, but a polypeptide close to the predicted size (20.4 kDa, including the His tag) was readily isolated from this fraction by Ni2+ affinity and anion exchange chromatography (Fig. 4A). The purified YlgG constituted ~0.2% of total soluble protein.



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  		FIG. 3.
Alignment and phylogenetic analysis of the deduced amino acid sequences of YlgG and selected Arabidopsis Nudix proteins. A, alignment of YlgG (GenBankTM AAN64308 [GenBank] with its closest Arabidopsis homolog, the At1g68760 gene product. Identical residues are shaded in black, and similar residues are shaded in gray. Dashes are gaps introduced to maximize alignment. The Nudix motif is overlined. B, unrooted phylogram, generated using the neighbor-joining method and 1,000 bootstrap iterations of YlgG, the At1g68760 protein, and other Arabidopsis proteins sharing homology with YlgG outside the Nudix box. Asterisks show branch points with bootstrap values >70%. Phylogenetic analysis was carried out using PHYLIP at the Institut Pasteur server (bioweb.pasteur.fr). Sequences were aligned using Multalin (prodes.toulouse.inra.fr/multalin/multalin.html).

 



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  		FIG. 4.
Isolation, reaction products, and Mg2+ response of recombinant YlgG. A, SDS-PAGE of soluble proteins from E. coli BL21-CodonPlus®-RIL cells induced by isopropyl-D-thiogalactopyranoside (lane 1) and YlgG protein purified by Ni2+-affinity and anion exchange chromatography (lane 2). The gel was stained with Coomassie Blue. Positions of molecular mass standards (kDa) are marked. B, analysis of the products of the action of purified YlgG on DHNTP. Reactions were run for 10 min in the standard assay conditions in the presence of 1 mM Mg2+, 100 mM KCl, and 0.1 mM DHNTP. DHNP was quantified by fluorometric HPLC. Pyrophosphate was determined as phosphate after treating with pyrophosphatase. Data are means of three determinations and S.E. C, response of DHNTP pyrophosphohydrolase activity to Mg2+. Assays contained 0.2 mM DHNTP and 100 mM KCl. Data are means of three determinations ± S.E.

 
Enzyme assays showed that purified YlgG had high DHNTP pyrophosphohydrolase activity, i.e. that it cleaved DHNTP to give DHNP and pyrophosphate in a 1:1 ratio, with negligible production of free phosphate (Fig. 4B). This activity was dependent on Mg2+ and reached ~70% of its maximal value at 1 mM (Fig. 4C). Because 1 mM is approximately the physiological level of free Mg2+ (11, 12), this concentration was adopted for subsequent work. Ca2+ at 1 mM did not activate YlgG; Zn2+ and Mn2+ could not be tested as they caused rapid chemical breakdown of DHNTP (9). At 1 mM Mg2+, chemical conversion of DHNTP to DHNP was almost undetectable (<0.5% of the maximal rate of the enzymatic reaction in Fig. 4C). This chemical conversion increased when Mg2+ or Ca2+ concentration was raised to 10–12 mM as reported (10), but at these concentrations other major breakdown products were also formed (not shown).

Kinetic characterization of YlgG (Table IV) gave a Km value for DHNTP (2.1 µM) that is among the lowest reported for Nudix enzymes and gave a fairly typical kcat value (0.21 s–1 at 30 °C) (34). YlgG had no detectable activity with GTP, ATP, TTP, CTP, the corresponding deoxynucleoside triphosphates, 8-oxo-dGTP, NADH, NAD+, diadenosine tri- or tetraphosphate, ADP-ribose, UDP-glucose, or coenzyme A (the detection limit was ≤1% of the activity with DHNTP). Consistent with this lack of activity, the YlgG amino acid sequence (Fig. 3A) does not have the motifs associated with specificity for NADH, diadenosine polyphosphates, ADP-ribose, or coenzyme A (35–38). The native molecular mass of recombinant YlgG was estimated to be 24.2 kDa on the basis of its elution from a calibrated size exclusion column. This value is close to the calculated mass of the His-tagged polypeptide (20.4 kDa), indicating that the native enzyme is a monomer.


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  		TABLE IV
Kinetic constants of YlgG with DHNTP as substrate

Values were determined at 30 °C in HEPES-KOH buffer, pH 8.0, containing 100 mM KCl and 1 mM Mg2+. Data are means of three independent determinations ± S.E.

 
Arabidopsis Homologs of YlgG—BLAST searches of the Arabidopsis genome revealed 20 proteins with a canonical Nudix motif (see supplemental Table I). Most of these share homology with YlgG only in the area of the Nudix box, but five (shown in Fig. 3B) have some degree of overall homology. The overall homology is greatest for the At1g68760 gene product, which is 34% identical and 44% similar to YlgG in a 73-residue region spanning the Nudix box (Fig. 3A). Phylogenetic analysis confirmed that this protein is more closely related to YlgG than the other four (Fig. 3B). Moreover, the At1g68760 protein is of similar size (16.4 kDa) to YlgG and, like other early enzymes in plant folate biosynthesis (4, 6), has no obvious organellar targeting sequence. Like YlgG, the At1g68760 protein lacks the motifs associated with preference for NADH, diadenosine polyphosphates, ADP-ribose, or coenzyme A (35–38). Finally, the At1g68760 protein is by far the closest Arabidopsis homolog (33% identity, 51% similarity) of the Nudix moiety of a predicted Parachlamydia fusion protein (GenBankTM YP008156) whose other domain is the folate synthesis enzyme dihydroneopterin aldolase (FolB). (This fusion arrangement strongly implies that the Nudix domain is the Parachlamydia DHNTP pyrophosphohydrolase.) Several lines of bioinformatic evidence thus point to the At1g68760 gene product as the best candidate for DHNTP pyrophosphohydrolase in Arabidopsis.

Characterization of the At1g68760 Gene Product—The At1g68760 gene product with or without a C-terminal His tag was abundantly expressed in E. coli (Fig. 5A). Pilot assays with desalted extracts indicated that the tag made no difference to the enzymatic characteristics of the protein, so all further work was done on the tagged protein after purification to homogeneity by Ni2+ affinity chromatography (Fig. 5A). Like YlgG, the At1g68760 protein had DHNTP pyrophosphohydrolase activity, producing DHNP and pyrophosphate in a 1:1 ratio but essentially no free phosphate (Fig. 5B). Also like YlgG, activity required Mg2+ (Fig. 5C), and this could not be replaced by Ca2+ (1 mM). Unlike YlgG, however, in the presence of 1 mM Mg2+ the At1g68760 protein was far more active against dGTP, 8-oxo-GTP, dTTP, and other nucleoside and deoxynucleoside triphosphates than against DHNTP (Table V). Activities with other Nudix substrates were negligible (Table V).



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  		FIG. 5.
Isolation, reaction products, and metal responses of recombinant At1g68760 protein. A, SDS-PAGE of soluble proteins from E. coli BL21-CodonPlus®-RIL cells induced by isopropyl-D-thiogalactopyranoside (lane 1) and the At1g68760 protein purified by Ni2+-affinity chromatography (lane 2). The gel was stained with Coomassie Blue. Positions of molecular mass standards (kDa) are marked. B, analysis of the products of the action of the purified protein on DHNTP. Reaction conditions are as shown in Fig. 4 except that incubation time was 30 min and DHNTP concentration was 0.3 mM. Data are means of three determinations and S.E. C, response of DHNTP pyrophosphohydrolase activity to Mg2+ concentration. Assays contained 50 mM HEPES-KOH, pH 8.0, 100 mM KCl, and 300 µM DHNTP. Data are means of three determinations ± S.E. D, response of NADH pyrophosphatase activity to Mn2+ or Mg2+ concentration. Assays were carried out in 50 mM Tris-HCl, pH 8.0, in the presence of 1 mM dithiothreitol and 2 mM NADH (21). Data are means of three determinations ± S.E. Note that values for Mg2+ are multiplied x100. Where no error bars are shown in C and D, they were smaller than the symbols.

 


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  		TABLE V
Substrate preference of the At1g68760 protein

Substrate concentrations were 0.5 mM. Activities were determined at 30 °C in HEPES-KOH, pH 8.0, containing 100 mM KCl and 1 mM Mg2+.

 
To further explore this striking contrast with the substrate specificity of YlgG, we evaluated the kinetic properties of the At1g68760 protein with DHNTP or dGTP as substrate in the presence of 1 mM Mg2+ (Table VI). The Km value for DHNTP was fairly similar to that for dGTP, but the kcat value was 500-fold lower, so that the catalytic efficiency was far higher for dGTP.


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  		TABLE VI
Kinetic constants of the At1g68760 protein (AtNUDT1)

Values were determined at 30 °C in HEPES-KOH buffer, pH 8.0, containing 100 mM KCl and 1 mM Mg2+. Data are means of three independent determinations ± S.E.

 
The At1g68760 gene product was reported by Dobrzanska et al. (21) to have high NADH pyrophosphatase activity and was designated AtNUDT1 (we will use this name henceforth). This activity against NADH was, however, characterized using an Mn2+ concentration of 5 mM, which is ~103-fold greater than typical levels of Mn2+ in growing cells (39). Low activity was also found in the presence of 5 mM Mg2+ (21). We therefore measured NADH pyrophosphatase activity at various Mn2+ and Mg2+ concentrations (Fig. 5D). This activity fell drastically as Mn2+ concentration approached the usual physiological range, and at 5 µM Mn2+ was only 0.001% that at 5 mM (Fig. 5D). With Mg2+, activity was insignificant except at the highest concentrations tested (Fig. 5D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our genetic and biochemical results with L. lactis identify the YlgG protein as a specific DHNTP pyrophosphohydrolase that participates in the folate pathway. We therefore propose that the gene encoding this protein be redesignated folQ. The Mg2+ requirement, substrate specificity, Km value, and native molecular mass of YlgG are very like those reported for the partially purified DHNTP pyrophosphohydrolase of E. coli (9). As E. coli and L. lactis are not closely related, the occurrence of similar DHNTP pyrophosphohydrolases in both species implies that specific DHNTP-cleaving Nudix enzymes are common to a wide range of bacteria.

Although deleting ylgG reduced folate production markedly in L. lactis, it did not eliminate it and did not affect growth. The residual folate synthesis in the deletion mutant is not unexpected, because broad spectrum phosphatases can attack DHNTP (40), and the massive accumulation of DHNTP seen in the mutant could potentially drive significant flux through nonspecific, normally minor, enzymatic dephosphorylation reactions. The accumulation of dihydroneopterin diphosphate found in the YlgG-deficient cells supports this possibility, for it indicates that iterative removal of single phosphate residues has inefficiently replaced removal of pyrophosphate as the main route from DHNTP to DHNP.

Two lines of evidence show that metal ion-catalyzed chemical dephosphorylation of DHNTP (10) is certainly not a major process under normal in vivo conditions and is probably not even a significant minor one. First, the large build up of DHNTP in deletant cells indicates that most of the DHNTP -> DHNP flux is normally mediated by YlgG. Second, the rate of chemical dephosphorylation was negligible in in vitro assays made at physiological pH and Mg2+ levels. The proposal that chemical dephosphorylation is important (10) was based solely on biochemical experiments involving unphysiological Mg2+ or Ca2+ levels and is unsupported by genetic or other in vivo evidence.

Cleavage of pyrophosphate from DHNTP is a new addition to the list of reactions shown to be mediated by Nudix enzymes (19, 36, 41, 42). The Mg2+ response observed for YlgG is very similar to those of Nudix nucleoside triphosphate pyrophosphohydolases such as E. coli MutT (the archetype of this enzyme class), which requires two divalent cations, one coordinated by the nucleoside triphosphate and the other by conserved residues in the Nudix motif and elsewhere (43). The low Km value observed (2.1 µM) for YlgG is consistent with the estimated in vivo concentration of DHNTP in wild type L. lactis cells (0.33 µM).

It is intriguing that the DHNTP concentration in wild type L. lactis is 10-fold higher than that of DHN, because this suggests that the rate of DHNTP dephosphorylation limits the pool size of DHN, and hence flux from DHN to folates. If this is the case, increasing YlgG expression could potentially enhance folate production in L. lactis strains engineered to overproduce DHNTP (15).

DHNTP is an intermediate in the biosynthetic pathway of tetrahydrobiopterin as well as of folate, and organisms such as pseudomonads, cyanobacteria, and fungi have both pathways (44–47). If these organisms use a DHNTP pyrophosphohydrolase, then this enzyme would be the one that commits pteridine moieties to folate synthesis. This differs from the usual situation in which the committing role is played by GTP cyclohydrolase I, the enzyme that produces DHNTP (1, 10). Regardless of whether DHNTP pyrophosphohydrolase has a committing role, it would seem to be a poor target for antibacterial drug discovery because inactivating it, in L. lactis at least, did not totally suppress folate synthesis and had no impact on growth.

The previously known bacterial folate synthesis enzymes all have homologs in plants (2–7), and the same is true for YlgG, although the homology in this case is weaker than usual. More generally, bacterial Nudix enzymes often share sequence features outside the Nudix box with eukaryotic enzymes having the same activity (35, 38, 47, 48). This pattern of cross-kingdom structural similarity between functionally equivalent Nudix hydrolases in itself makes the original identification of At-NUDT1 as an NADH pyrophosphatase (21) seem suspect, for this protein lacks the SQPWPFPXS motif found downstream of the Nudix box in other NADH pyrophosphatases (35, 38). (This motif is, however, present in another Arabidopsis protein, the At5g20070 gene product that has yet to be characterized, see Ref. 38.) This suspicion about the function of AtNUDT1 was corroborated by biochemical evidence, for we found that the activity against NADH requires unphysiological levels of Mn2+ and that DHNTP and nucleoside and deoxynucleoside triphosphates are far better substrates than NADH in the presence of physiological Mg2+ levels. A similar switch in substrate preference when Mg2+ replaces Mn2+ has been reported for another Nudix hydrolase (49).

The catalytic efficiency (kcat/Km) of AtNUDT1 for DHNTP is ~104-fold lower than that of YlgG and, unlike YlgG, AtNUDT1 is not at all specific for DHNTP, deoxynucleoside and nucleoside triphosphates being much better substrates. These findings, added to the probability that cytosolic deoxynucleoside and nucleoside triphosphate levels greatly exceed that of DHNTP (17, 50), raise the question of whether AtNUDT1 could account for DHNTP hydrolysis in vivo or whether another, more efficient, DHNTP pyrophosphohydrolase remains to be discovered. We cannot at this point exclude the existence of another enzyme, but two arguments suggest there is no need to invoke one. First, the DHNTP -> DHNP flux is so small that it might well be sustained even by an inefficient enzyme operating in the presence of a large excess of better substrates. (This flux can be estimated from data on folate turnover in plants as ≤15 pmol h–1 g–1 fresh weight (51, 52), which is ~106-fold smaller than the respiratory ADP -> ATP flux (53), for example.) Second, there are precedents for Nudix hydrolases mediating key minor reactions while awash in alternative substrates that they are concurrently cleaving. MutT-type Nudix enzymes are thought to sanitize nucleotide pools by removing oxidized, mutagenic derivatives such as 8-oxo-dGTP. These enzymes usually prefer the oxidized derivatives but, like At-NUDT1, also attack normal deoxynucleoside and nucleoside triphosphates, which are far more abundant in vivo (54–57). Because the substrate range of AtNUDT1 is like that of MutT-type hydrolases (54), it could be a bifunctional DHNTP pyrophosphohydrolase/sanitizing enzyme. If so, the metabolic inefficiency involved in hydrolyzing large amounts of normal nucleotides as well as DHNTP would add no extra energy cost because such wastage would already be part of the vital sanitizing function of the enzyme.


   FOOTNOTES
 
* This work was supported in part by the Florida Agricultural Experimental Station, by an endowment from the C. V. Griffin, Sr., Foundation, and by Grant 2005-35318-15228 from the National Research Initiative Competitive Grants Program of the United States Department of Agriculture. This work was approved for publication as Journal Series number R-10630. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: Horticultural Sciences Dept., University of Florida, P. O. Box 110690, Gainesville, FL 32611. Tel.: 352-392-1928; Fax: 352-392-5653; E-mail: adha{at}mail.ifas.ufl.edu.

1 The abbreviations used are: DHNTP, 7,8-dihydroneopterin triphosphate; DHNP, 7,8-dihydroneopterin monophosphate; NMNH, reduced nicotinamide mononucleotide; HPLC, high pressure liquid chromatography; DHN, dihydroneopterin; X-gal, 5-bromo-4-chloro-3-indoyl-galactopyranoside. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Markus Fischer and Adelbert Bacher for the gift of recombinant E. coli GTP cyclohydrolase I.



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