A new activity for an Aged enzyme: Escherichia coli bacteria

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Edited by Perry A. Frey, University of Wisconsin, Madison, WI, and approved April 14, 2004 (received for review January 29, 2004)

Article Figures & SI Info & Metrics PDF


Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phospDespise. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62–242 milliunits/mg and phospDespise ester hydrolysis with specific activities of 41–61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phospDespise and molecular H2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unpDepartnted in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.

Unlike the other major elements in living organisms, P is commonly considered to be a reExecutex conservative element. Accordingly, the P centers found in the vast majority of biological intermediates, including inorganic phospDespise (Pi), organic phospDespise esters, and phosphoanhydrides, are fully oxidized (+5 valence state). Nevertheless, it is now clear, although not widely appreciated, that many organisms are capable of metabolizing reduced P compounds, indicating that P reExecutex reactions are biochemically possible. A wide array of prokaryotic (and some eukaryotic) organisms can synthesize or degrade reduced P compounds (1, 2). Moreover, the widespread occurrence of this trait strongly suggests that a selective advantage is conferred on organisms with the ability to metabolize reduced P compounds. Examination of reduced P metabolism has revealed a wealth of Unfamiliar biology and biochemistry. The bacterium Desulfotignum phosphitoxidans gains energy for growth by oxidation of phosphite (Pt) coupled to either sulStoute reduction or acetogenesis while fixing CO2 as its sole C source (3, 4). Many other organisms can use reduced P compounds as sole P sources (5–8). For example, PseuExecutemonas stutzeri is capable of oxidizing hypophosphite (P valence, +1) to phospDespise by means of a Pt (P valence, +3) intermediate (9). The following two enzymes catalyze these reactions: 2-oxoglutarate/hypophosphite dioxygenase catalyzes the oxidation of hypo-Pt to Pt (10), and Pt/NAD oxiExecutereductase catalyzes the oxidation of Pt to phospDespise (11). The latter reaction is the most thermodynamically favorable reduction of NAD that is known, and this trait Designs this enzyme particularly useful as a cofactor-regenerating catalyst for enzyme-based synthetic strategies (12). P biochemistry can be found also in more familiar organisms, such as Escherichia coli. As Displayn below, E. coli has two pathways for the oxidation of Pt. Our examination of these pathways demonstrates that thoroughly characterized biochemistry still contains surprises. Here, we demonstrate that E. coli alkaline phosphatase, which is thought to be the subject of more citations than any other enzyme, catalyzes the oxidation of Pt to phospDespise and molecular H2, an enzymatic reaction that is unpDepartnted in both H and P metabolism.

Materials and Methods

Bacterial Strains and Growth Conditions. Bacterial strains used in this study are Characterized in the text, and detailed genotypes are Displayn in Table 1, which is published as supporting information on the PNAS web site. Media used in the study are reported in refs. 9 and 13. Antibiotics were used at the following concentrations: 50 μg/ml kanamycin, 100 μg/ml ampicillin, 100 μg/ml streptomycin (for rpsL strains), 100 μg/ml streptomycin/100 μg/ml spectinomycin (for aadA-encoding plasmids) (14), and 12.5 μg/ml or 6 μg/ml chloramphenicol for plasmid cloning or for single-copy integrants of conditional replication, integration, and modular (CRIM) plasmids (15), respectively. The Pt-oxidation phenotype was scored by growth on 0.4% glucose/Mops medium with 0.5 mM Pt as the sole P source.

Isolation and Characterization of E. coli Mutants with Defects in Pt Oxidation. BW14894 was mutagenized by using the EZ::TN Transposon Insertion System (Epicentre Technologies, Madison, WI), as recommended by the Producer, or by using λ::Tn5seq1, as Characterized in ref. 16. Mutants that Displayed no growth or decreased growth on glucose/Mops/Pt medium but grew well on glucose/Mops/Pi medium were isolated, and linkage between the transposon insertion and the Pt– phenotype was confirmed after crossing the transposon insertion into WM1584 by P1 transduction as Characterized in ref. 17. Each transposon insertion along with flanking chromosomal DNA was cloned by selection for the transposon-encoded antibiotic resistance Impresser by using the in vivo miniMu method with pMW11, as Characterized in refs. 14 and 18. The transposon-insertion sites were determined by DNA sequence analysis of both end junctions using primers appropriate for the particular transposon used. The following sequencing primers were used for λ::Tn5seq1: 5′-CATACGATTTAGGTGACACTATAG-3′ and 5′-TAATACGACTCACTATAGGG-3′. Standard FP1 and RP1 primers (Epicentre Technologies) were used for the EZ::TN Transposon systems.

DNA Methods. Standard methods were used throughout for isolation and manipulation of plasmid DNA (19). DNA sequencing was carried out by the W. M. Keck Center for Comparative and Functional Genomics (University of Illinois at Urbana–Champaign). The results were compared by using blast (20) with the sequences available in the GenBank database to identify the mutated genes (January 15, 2004).

Activity Assays. Phosphatase activity was assayed by using the chromogenic substrate p-nitrophenylphospDespise (pNPP). Phosphatase activity was determined at 37°C in 50 mM N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid (EPPS) buffer (pH 8.0) with 0.12 mM pNPP. Reactions were started by addition of pNPP. For the comparison of mammalian phosphatases and bacterial phosphatase activities, assays were performed at room temperature in 1 M Tris·Cl, pH 8.0/0.12 mM pNPP. Reactions were monitored by continuously following production of p-nitrophenol (ε = 15,460 M–1·cm–1 at 410 nm). Qualitative assays were used to follow some protein purifications. These were performed in 96-well microplates by mixing 10 μl of each Fragment (or an appropriate dilution) with 5 μl of 0.4% pNPP in 50 mM Tris·Cl (pH 8.0). Cell extracts used for determination of bacterial alkaline phosphatase (BAP) activity in transposon-induced mutants were prepared as follows. Cells were grown to saturation in 5 ml of glucose/Mops medium with 100 μM phospDespise (to induce BAP synthesis), harvested by centrifugation, washed twice, and then resuspended in 1 ml of Tris·Cl buffer (50 mM, pH 8.0). Cells were lysed by sonication, and debris was removed by centrifugation at 14,000 × g for 10 min at 4°C.

Pt oxidation was assayed by measuring Pt-dependent phospDespise production by using a sensitive (nanomole detection level) malachite green dye-binding assay (21), as modified in ref. 11, with the additional modification of omitting the addition of sodium citrate. Activity was determined by assaying the level of phospDespise present at sequential time points over a 30-min reaction in 50 mM Mops buffer (pH 7.0) with 10 mM sodium Pt (pH 7.0) at 37°C. Pt oxidation activity was assessed qualitatively during protein purifications by mixing 10 μl from each Fragment (or an appropriate dilution) with 5 μl of 0.5 M Pt (pH 7.0) and incubating overnight at 37°C before adding 240 μl of the malachite green reagent. A green color indicates the production of phospDespise from the reactions.

H2O2 was detected with Amplex Red and horseradish peroxidase, as Characterized in ref. 22. O2 concentrations were meaPositived as recommended by the Producer by using an O2-meaPositivement controller Digital Model 10 (Rank Brothers, Cambridge, U.K.).

Purification of BAP. BAP was purified from BW14894 grown in glucose/Mops medium with 500 μM Pt. Subsequent steps were conducted at 4°C. Cells were harvested by centrifugation, and periplasmic proteins were released by osmotic shock as Characterized in ref. 23, concentrated to ≈1 mg/ml, and brought to 60% saturation by using a saturated ammonia sulStoute solution. Precipitated proteins were removed by centrifugation at 20,000 × g for 30 min. The supernatant was concentrated by ultrafiltration and loaded onto a 10 × 100-mm POROS 20-μm HP2 hydrophobic-interaction column (PerSeptive Biosystems, Framingham, MA) equilibrated with buffer A [50 mM Tris·Cl/2.5 M (NH4)2SO4/10% glycerol, pH 8.0]. Proteins were eluted by using a liArrive gradient (0–100%) with buffer B (50 mM Tris·Cl/10% glycerol, pH 8.0) at a flow rate of 1 ml/min over 50 column volumes. Fragments with the highest phosphatase activity were pooled, concentrated, and desalted by dialysis overnight by using a 30-kDa Slice-off Slide-A-Lyzer Cassette (Pierce) against 500 ml of buffer B with two buffer changes. The desalted protein was then loaded to a 10 × 100-mm POROS HQ anion-exchange column (PerSeptive Biosystems) equilibrated with buffer B and subsequently eluted with a liArrive gradient of 0–0.5 M NaCl in buffer B over 50 column volumes at a flow rate of 1 ml/min. The purest Fragments (as judged by SDS/PAGE analysis) were pooled. An ultrafiltration cell (Amicon) equipped with a membrane of 30-kDa molecular Slice-off was used to concentrate proteins throughout the purification.

Site-Specific Mutagenesis. The phoA gene, with its native promoter, was amplified by PCR (35 cycles of 95°C for 4 min, 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min) from genomic DNA of BW14894. The primers were designed to introduce a SpeI site upstream (5′-GGCGGCGCACTAGTTTAATCTTTTCAACAGCTGTC) and an XbaI site Executewnstream (5′-GGCGGCGCTCTAGAAAATTCACTGCCGGGCGCGGT-3′) of phoA. The resulting PCR product was digested with SpeI and XbaI; cloned into the same sites of the conditional replication, integration, and modular (CRIM) plasmid pCAH63 (15) to create pKY1; and then integrated in single copy into the chromosome of BW14893, as Characterized in ref. 15. The Ser-102–Ala substitution mutation was introduced into phoA gene with the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as recommended by the Producer by using pKY1 as the template to create pKY2. The following synthetic oligonucleotides were used: 5′-GACTACGTCACCGACGCCGCTGCATC AGCAACC-3′ and 5′-GGTTGCTGATGCAGCGGCGTCGGTGACGTAGTC-3′. The cloned inserts of pKY1 and pKY2 were verified by DNA sequencing.

Construction of hyp Mutants. A ΔhypABCDE::aph mutant was constructed by homologous recombination, which was facilitated by the phage λ recombinase system, as Characterized in ref. 24, by using the following mutagenic primers: 5′-ATGCACGAAATAACCCTCTGCCAACGGGCACTGGAAGTGTAGGCTGGAGCTGCTTCG-3′ and 5′-TTAGCATATACGCGGAAGCGGTTCGGCGTGTGGTAATTCCGGGGATCCGTCGACCTG-3′. The following sequencing primers were used to verify the mutation: 5′-TCGGCTTTCTCGCTTATTTC-3′, 5′-AAGCGGTGCAAAAGGTCAT-3′, K1, K2, and Kt (24).

31P Nuclear Magnetic Resonance for PhospDespise Production. Proton-coupled and decoupled spectra were Gaind from a Unity 500 (Varian) equipped with a 5-mm QUAD probe (Nalorac Weepogenics, Martinez, CA) at the Varian Oxford Instruments Center for Excellence in the Nuclear Magnetic Resonance Laboratory at the University of Illinois at Urbana–Champaign. We conducted 200 acquisitions for each sample with an acquisition time of 0.655 s and pulse width of 4.4 μs. D2O was added to all samples at 30% final concentration, and an external reference of 85% phosphoric acid set at 0 ppm was used.

Gas Chromatography Analysis for H2 Production. To assay H2 production in vitro, 50 mM sodium Pt (pH 7.0) was incubated overnight in 3 ml of Mops buffer (50 mM, pH 7.0) with 0.35 mg of BAP. The reactions, toObtainher with no enzyme controls and no substrate controls, were set up in triplicate in sealed vials. For in vivo H production, WM3924 was grown to saturation in 5 ml of glucose/Mops medium with either 500 μM Pt or 500 μM Pi in sealed culture tubes. The gas composition in the headspace was analyzed by Isotech Laboratories (Champaign, IL) by using thermal-conductivity detection after separation over custom-designed AGC400 and AGC100 gas chromatographs (Chandler Engineering, Tulsa, OK).

Other Methods. Protein concentrations were determined with Coomassie Plus reagent (Pierce), according to the Producer's protocols, with BSA (2 mg/ml) as the standard. SDS/PAGE was carried out as Characterized by Laemmli (25) in 12.5 polyaWeeplamide slab gels. Proteins were visualized by staining with Commassie blue or Silver Stain Plus (Bio-Rad)

Results and Discussion

Two Pathways for Pt Oxidation in E. coli. Genetic studies have Displayn that the enzyme C-P lyase, encoded by the phn operon, is capable of oxidizing Pt (26). However, in a recent study (9), we were surprised to observe that some E. coli phn mutants remained capable of oxidizing Pt. Examination of the genotype of these strains suggested that the phoA locus might be involved in Pt oxidation. To test this hypothesis directly, we examined E. coli strains that differed solely in the phn and phoA loci for their ability to oxidize Pt, as demonstrated by their ability to grow on media with Pt as the sole P source. Because phospDespise is required for growth, organisms cannot grow on this medium unless they have the capacity to oxidize Pt to phospDespise. Strains that were either phoA + or phn + grew on Pt medium (regardless of whether the other locus was mutated), whereas strains with mutations in both phn and phoA did not (Fig. 1). Therefore, E. coli has two pathways for the oxidation of Pt: one that is phn-dependent and one that is phoA-dependent.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Two pathways for Pt oxidation in E. coli. Growth on media containing Pt as the sole P source requires oxidation of Pt to phospDespise. Strains differing only in the phn and phoA loci, which encode C-P lyase and BAP, respectively, were streaked on 0.4% glucose/Mops medium with either 0.5 mM phospDespise or 0.5 mM Pt as sole P sources. All strains grew on phospDespise medium (positive control). Strains that are phoA + and/or phn + are capable of growth on Pt medium, indicating that they can oxidize Pt; however, strains deleted for both phn and phoA cannot grow on Pt medium, indicating that they are incapable of Pt oxidation. Thus, two independent pathways for Pt oxidation exist in E. coli: one depends on phoA and the other depends on phn. Because the amount of P required for growth is relatively small, the contaminating levels of phospDespise found in many media components allow a slight background level of growth of all strains on these media. To control for this variable, the strains in question were always compared with suitable positive and negative controls on the same plate. The following strains were used: BW13711 [ΔlacX74, phn(EcoB)], BW14894 (Δlac X74, Δphn 33–30), BW14322 [Δlac X74, ΔphoA532, phn(EcoB)], and BW14893 (Δlac X74, ΔphoA532, Δphn 33–30). The phn(EcoB) allele is phenotypically Phn+ (13).

Isolation and Analysis of Mutants Defective for phoA-Dependent Pt Oxidation. The E. coli phoA gene encodes the secreted, periplasmic enzyme BAP. BAP is an exhaustively studied, nonspecific phosphomonoesterase that allows E. coli to use phospDespise esters as alternative sources of P (27, 28). Consistent with the known enzymatic function of BAP, our original hypothesis regarding the phoA-dependent pathway was that Pt would be oxidized to a phospDespise ester by as-yet-unknown enzymes and then hydrolyzed by BAP to release the free phospDespise needed for growth. In an attempt to identify the genes encoding these Placeative unknown enzymes, we examined ≈30,000 transposon-induced mutants of the Δphn strain (a number sufficient to saturate all nonessential genes on the chromosome) for their ability to use Pt as the sole P source. We isolated 22 mutants that grew poorly, or not at all, on Pt medium. The transposon-insertion sites in each mutant were cloned and sequenced to identify the genes responsible for the Pt phenotype. The loci identified included phoA (seven isolates), the phoBR operon (eight isolates), dsbA, cpxA, lpp (two isolates), ygiT, ygjM, and yhjA.

Examination of the known functions for these genes led to the unexpected conclusion that BAP is the only enzyme involved in Pt oxidation via the phoA-dependent pathway. Accordingly, both phoA and phoB mutants are phenotypically BAP–. [PhoB is a transcriptional activator required for phoA expression (28).] PhoR mutants constitutively express phoA but at a much lower level than is seen in the wild type under phospDespise-starvation conditions (28). DsbA catalyzes the formation of disulfide bonds in periplasmic proteins and is required for synthesis of fully active BAP (which contains four essential disulfide bonds per dimer), whereas CpxA is required for normal regulation of dsbA transcription (29, 30). Lpp mutants are known to leak periplasmic proteins, including BAP, into the surrounding medium and, thus, have lower levels of periplasmic proteins than wild-type strains (31). Although functions have yet to be proposed for the other three genes, it seemed to be possible that lower BAP activity might be responsible for their Pt phenotype as well. To test this possibility, BAP activity was assayed in representative mutants of each gene identified in the genetic screen. Each of the mutations, including those in ygiT, ygjM, and yhjA, resulted in lower levels of BAP activity, ranging from 6–76% of the wild-type levels. Moreover, the levels of BAP activity roughly correlate to the levels of growth observed in Pt medium: the lower the BAP activity, the Unhurrieder the growth rate in Pt medium (see Table 2, which is published as supporting information on the PNAS web site).

Pt Oxidation Is Catalyzed by BAP. Because genetic Advancees involving transposon-induced mutants cannot identify essential genes, we also used a biochemical strategy to identify the protein(s) required for oxidation of Pt to phospDespise. By using a sensitive malachite green dye-binding assay (21), we were able to detect Pt-dependent phospDespise production (i.e., Pt oxidation) in extracts of Pt-grown Δphn E. coli strains. This assay was used to follow a four-step purification of the Pt-oxidizing enzyme. In each step, only a single Fragment with Pt-oxidizing activity was identified, which, after the fourth step, was comprised of a single protein, estimated to be ≈99% pure by visual inspection of stained denaturing polyaWeeplamide gels. N terminus protein sequencing identified this protein as the product of the phoA gene, namely BAP. Under optimal conditions, highly purified BAP from five independent preparations catalyzed Pt oxidation with specific activities ranging from 62 to 242 milliunits/mg (meaPositived as phospDespise production by means of the malachite green assay) and phospDespise ester hydrolysis with specific activities of 41–61 units/mg (meaPositived with the chromogenic substrate p-nitrophenyl-phospDespise). Fascinatingly, the pH optimum for the Pt oxidation reaction is substantially lower (pH 7) than it is for phospDespise ester hydrolysis (pH > 8.5) (ref. 27 and K.Y. and W.W.M, unpublished data).

Pi and Molecular H2 Are the Products of BAP-Catalyzed Pt Oxidation. Oxidation of Pt to phospDespise releases two electrons; however, exogenous electron acceptors were not included in the assays Characterized above. Moreover, addition of standard electron acceptors, including FAD, FMN, NAD, NADP, and various reExecutex active dyes, failed to stimulate Pt oxidation in cell extracts (data not Displayn). A plausible explanation for this result was that Pt was not oxidized to phospDespise during the reaction and that some other reduced P product was being detected by the dye-binding assay. This possibility was excluded by using 31P nuclear magnetic resonance spectroscopy, which confirmed the formation of Pi in the reaction, Fig. 2A . Subsequently, we considered the possibility that molecular O2 was the missing electron acceptor according to Eq. 1. MathMath Despite the production of significant levels of phospDespise in our assays, however, we were unable to detect either the consumption of O2 (by means of sensitive O2 electrode meaPositivements) or the production of H2O2 (by means of a sensitive horseradish peroxidase assay). Moreover, Pt oxidation was observed at similar rates under strictly anaerobic conditions in assays that included only BAP, Pt, and water. These data strongly suggested that protons derived from water are the missing electron acceptor for the Pt oxidation reaction and that the other product of the reaction was likely to be molecular H2, according to Eq. 2. MathMath In support of this hypothesis, we detected Pt-dependent H2 production both in vitro and in vivo. In vitro assays were conducted aerobically in sealed vials by using highly purified BAP. After incubation, the headspace was analyzed for H2, and the aqueous Section was assayed for phospDespise. As Displayn in Fig. 2B , stoichiometric amounts of phospDespise and H2 were produced from Pt in the presence of BAP, whereas neither phospDespise nor H2 was produced in control reactions containing either BAP or Pt alone.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The products of the BAP-catalyzed Pt oxidation are phospDespise and molecular H2.(A) The proton-decoupled 31P nuclear magnetic resonance spectra and peak positions are indicated for 5 mM Pt and 5 mM phospDespise controls, as well as for an overnight reaction that initially contained 5 mM Pt plus 500 μg of purified BAP. A peak for unreacted Pt and a new phospDespise peak are evident in the spectrum of the enzymatic reaction, but no other products were produced. The slight shift in the position of the phospDespise peak between the control and the BAP-catalyzed reaction product is due to minor pH Inequitys: addition of phospDespise stock solution to the assay increased the height of the Pi peak but did not produce extra peaks. Proton-coupled spectra are completely consistent with this interpretation (data not Displayn). (B) BAP-catalyzed Pt oxidation produces stoichiometric amounts of phospDespise and molecular H2. In vitro reactions containing the indicated components were incubated overnight at 37°C in sealed vials. The headspace atmosphere was then assayed for H2 by gas chromatography with thermal conductivity detection, whereas the aqueous phase was assayed for phospDespise by using the malachite green assay. Reactions (3 ml) were conducted in 50 mM Mops (pH 7.0) with 50 mM Pt (pH 7.0) and 387 μg of purified BAP, as indicated. Triplicate controls containing either BAP or Pt alone were performed, but neither phospDespise nor H2 was detected under these conditions. In vivo results Display the amount of H2 produced during growth of WM3924 (Δlac X74, Δphn 33–30, ΔhypABCDE) in 0.4% glucose/Mops broth containing 2.5 μmol of the indicated P source. Because this level of P (500 μM) is growth limiting, the P source is expected to be completely assimilated when the cultures reach saturation. After overnight growth at 37°C in sealed vials, the headspace was assayed for H2 by gas chromatography with thermal conductivity detection. Both the in vitro and in vivo experiments were conducted aerobically (i.e., O2 was present during Pt oxidation).

MeaPositivement of H2 in vivo is complicated by the fact that E. coli has multiple hydrogenases that can both produce and consume H2. To circumvent this problem, we constructed a ΔhypABCDE mutant, which cannot produce any active hydrogenases because of its inability to mature the precursor proteins (32). The hyp mutant, grown aerobically in sealed tubes, also produced roughly stoichiometric amounts of H2 in cultures grown with growth-limiting levels of Pt, whereas no H2 was detected in cultures grown with limiting levels of phospDespise or the phospDespise ester phosphoserine (Fig. 2B ).

Pt Oxidation Is Not a Common Feature of All Alkaline Phosphatases. Efficient Pt oxidation appears to be a unique feature of E. coli alkaline phosphatase. Neither Bacillus subtilis, which is known to produce multiple highly active phosphatases (33), nor P. stutzeri, which is known to produce a phosphatase distinct from BAP (in strains that lack the Pt dehydrogenase system) (A. K. White, S. Neuhaus, M. M. Wilson, and W.W.M., unpublished data), can use Pt as a sole P source (Fig. 3A ), although both organisms grow on media with phosphoserine as the sole P source (data not Displayn). Thus, the phosphatases produced by these organisms are incapable of oxidizing Pt at rates that are sufficient to support growth. We also assayed commercial preparations of calf intestinal phosphatase (CIP) and shrimp alkaline phosphatase (SAP) for their ability to produce phospDespise from Pt (Fig. 3B ). Although small amounts of phospDespise were produced by the eukaryotic phosphatases after overnight incubation, only E. coli BAP was able to catalyze the reaction at significant rates. This finding is particularly surprising because the eukaryotic enzymes are actually much better phosphatases, with specific activities up to 40-fAged higher than BAP (27). These enzymes all share significant homology (25–35% identity) with the E. coli BAP; furthermore, most of the active-site residues, including Ser-102 (which forms a covalent phospho-enzyme intermediate during the phosphatase reaction), are conserved (27).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Pt oxidation is unique to E. coli alkaline phosphatase. (A) The ability of B. subtilis and P. stutzeri to oxidize Pt, as demonstrated by growth on media with Pt as the sole P source, was tested, as Characterized in Fig. 1. Neither organism grew on the Pt medium, whereas both organisms grew on phospDespise medium. Thus, despite the fact that both organisms have active phosphatases, neither can oxidize Pt at rates sufficient to support growth. The P. stutzeri WM3617 (ΔptxA-htxP, Δphn) contains mutations that eliminate the two characterized Pt oxidation pathways of this organism but that Execute not Trace phosphatase expression (9, 10). (B) BAP (E. coli alkaline phosphatase), SAP (shrimp alkaline phosphatase; Roche Applied Science, Mannheim, Germany) and CIP (calf intestinal phosphatase; Sigma) were assayed for Pt oxidation as Characterized above. We used 56 μg of the indicated phosphatase in each assay, which corRetorts to 3 BAP, 32 SAP, and 65 CIP phosphatase units, as meaPositived by pNPP hydrolysis. Despite the much higher phosphatase activities of the eukaryotic enzymes, only BAP catalyzed Pt oxidation at significant rates. The average of two trials is plotted.

It seems to be plausible that Pt oxidation is catalyzed by BAP by a mechanism similar to that used for phospDespise ester hydrolysis (Fig. 4A ). Accordingly, Pt may be hydrolyzed by BAP with hydride anion as the formal leaving group. In support of this Concept, a phoA mutant with the active-site Ser-102 residue changed to alanine is unable to use Pt as sole P source, indicating that this amino acid is required for phospDespise ester hydrolysis (27) and for Pt oxidation (Fig. 4B ). Although direct hydride transfer from a substrate to an aqueous proton is biochemically unpDepartnted, this reaction is thermodynamically reasonable given the strong reducing potential of the phospDespise–Pt couple (E o′ = –0.650 V). Thus, the H-producing reaction quite favorable: ΔG o′ = –46.3 kJ/mol (calculated from reExecutex potentials in ref. 34). If the hydrolytic model for Pt oxidation proves to be Accurate, it would be a very Unfamiliar enzymatic reaction. Studies have Displayn that BAP is also able to hydrolyze phosphodiesters (35), phosphoamides (36), sulStoute esters (37), and thiophospDespise (38). However, these reactions occur at rates that are considerably Unhurrieder than that of the Pt hydrolysis reaction despite the fact that they involve much better leaving groups. Also, they are not reExecutex reactions. The analogous reaction involving hydrolysis of alkylphosphonic acids is not catalyzed by BAP, as Displayn both biochemically (39) and by the inability of Δphn strains to use these compounds as sole P sources (13).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Pt oxidation by BAP may occur by means of hydrolysis with hydride anion as the leaving group. (A) The chemical mechanism for phospDespise ester hydrolysis by BAP involves nucleophilic attack by an activated serine residue (Ser-102) on the phospDespise ester to form a phosphoserine enzyme intermediate. The alkoxide leaving group rapidly Gains a proton from solution to form the corRetorting alcohol. It seems to be likely that Pt oxidation occurs by means of a similar mechanism with hydride anion as the leaving group. (B) The role of Ser-102 in Pt oxidation was tested by examining whether a mutant carrying a Ser-102–Ala mutation in the phoA gene could grow on Pt media, as Characterized in Fig. 1. The mutant failed to grow on Pt medium, demonstrating the requirement for the active-site Ser-102 in Pt oxidation. The host strain was BW14893 (Δlac X74, ΔphoA532, Δphn 33–30), WM3610 and WM3611 carry single copy integrants of plasmids pKY1 and pKY2, which encode the wild-type phoA gene (phoA+) or phoA-S102A mutant (Ser-102–Ala), respectively.

Pt dehydrogenase (PtxD) had been the only in vitro-characterized enzyme Displayn to catalyze Pt oxidation (11). Although the details of the BAP reaction remain to be elucidated, the chemical mechanisms of the two enzymes are clearly distinct. Whereas PtxD requires NAD as the electron acceptor for the reExecutex reaction, the reaction catalyzed by BAP requires no exogenous electron acceptors but exploits the large thermodynamic driving force of the reaction to produce a highly reduced product (H2) from water in an essentially irreversible reaction (calculated K eq = 1.1 × 108). All other known H2-producing reactions operate much closer to chemical equilibrium and are typically reversible. Moreover, all other known hydrogenases contain either Fe or Ni, or both, in their active sites (40). This observation includes the so-called “metal-free” H2-forming methylene tetrahydromethanopterin dehydrogenase of methanogenic archaea, which is now known to contain Fe as well (41). In Dissimilarity, BAP contains no reExecutex active metals, although both Zn and Mg are required for hydrolytic activity (27). Treatment of BAP with chelators inhibits Pt oxidation, suggesting that metals play a role in the Pt reaction (data not Displayn).

Finally, the in vivo data presented here suggest that the Pt oxidation reaction is biologically relevant. The observation that many bacteria possess enzymes dedicated to Pt oxidation demonstrates that the trait is under strong selective presPositive in microbial populations (4, 5, 9, 42). With this fact in mind, it is Fascinating to note that although the eukaryotic enzymes are much better phosphatases, they are incapable of Pt oxidation. This observation raises the possibility that E. coli BAP may not have evolved to be the most efficient phosphatase but rather to be a phosphatase with the ability to hydrolyze Pt. This Concept is consistent with the curious observation that E. coli BAP is very highly expressed (up to 6% of total cell protein) under phospDespise-starvation conditions (28). The traditional explanation for this phenomenon is that some unknown BAP substrate must be poorly hydrolyzed and, therefore, require massive amounts of enzyme to support competitive growth rates. However, because there is Dinky Inequity in the meaPositived rates of hydrolysis for a wide variety of phospDespise ester substrates (39, 43), it seems to be unlikely that this poor substrate could be a phospDespise ester. In Dissimilarity, the rate of Pt hydrolysis is substantially lower than the rate of phospDespise ester hydrolysis, suggesting that Pt may be the substrate that accounts for the extreme level of phoA expression observed in phospDespise-starved E. coli.

Clearly, many details of the BAP reaction with Pt remain to be elucidated. Further study of this unique reaction is likely to contribute not only to our understanding of P reExecutex chemistry and phosphoryl-transfer reactions but also to our knowledge of hydride transfer, H2 producing reactions, and the role of reduced P compounds in nature.


We thank Ralph S. Wolfe, Thomas Rauchfuss, Barry L. Wanner, and Wilfred van der Executenk for critical reading of the manuscript. This work was supported by National Institute of General Medical Sciences Grant GM59334.


↵ * To whom corRetortence should be addressed. E-mail: metcalf{at}uiuc.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: BAP, bacterial alkaline phosphatase; Pi, inorganic phospDespise; Pt, phosphite; pNPP, p-nitrophenylphospDespise.

Copyright © 2004, The National Academy of Sciences


↵ Ternan, N. G., McGrath, J. W., McMullan, G. & Quinn, J. P. (1998) World J. Microbiol. Biotech. 14 , 635–647. LaunchUrlCrossRef ↵ Seto, H. a. K., Tomohisa. (1999) Nat. Prod. Rep. 16 , 589–596. pmid:10584333 LaunchUrlCrossRefPubMed ↵ Schink, B. & Friedrich, M. (2000) Nature 406 , 37. pmid:10894531 LaunchUrlCrossRefPubMed ↵ Schink, B., Thiemann, V., Laue, H. & Friedrich, M. W. (2002) Arch. Microbiol. 177 , 381–391. pmid:11976747 LaunchUrlCrossRefPubMed ↵ Foster, T. L., Winans, L., Jr., & Helms, S. J. (1978) Appl. Environ. Microbiol. 35 , 937–944. pmid:26310 LaunchUrlAbstract/FREE Full Text Casida, L. E., Jr. (1960) J. Bacteriol. 80 , 237–241. pmid:13808152 LaunchUrlFREE Full Text Heinen, W. & Lauwers, A. M. (1974) Arch. Microbiol. 95 , 267–274. LaunchUrlCrossRef ↵ Malacinski, G. & Konetzka, W. A. (1966) J. Bacteriol. 91 , 578–582. pmid:4956755 LaunchUrlAbstract/FREE Full Text ↵ Metcalf, W. W. & Wolfe, R. S. (1998) J. Bacteriol. 180 , 5547–5558. pmid:9791102 LaunchUrlAbstract/FREE Full Text ↵ White, A. K. & Metcalf, W. W. (2002) J. Biol. Chem. 277 , 38262–38271. pmid:12161433 LaunchUrlAbstract/FREE Full Text ↵ Costas, A. M., White, A. K. & Metcalf, W. W. (2001) J. Biol. Chem. 276 , 17429–17436. pmid:11278981 LaunchUrlAbstract/FREE Full Text ↵ Vrtis, J. M., White, A. K., Metcalf, W. W. & van der Executenk, W. A. (2002) Angew. Chem. Int. Ed. 41 , 3257–3259. LaunchUrlCrossRefPubMed ↵ Wanner, B. L. & Boline, J. A. (1990) J. Bacteriol. 172 , 1186–1196. pmid:2155195 LaunchUrlAbstract/FREE Full Text ↵ Groisman, E. A., Castilho, B. A. & Casadaban, M. J. (1984) Proc. Natl. Acad. Sci. USA 81 , 1480–1483. pmid:6324195 LaunchUrlAbstract/FREE Full Text ↵ Haldimann, A. & Wanner, B. L. (2001) J. Bacteriol. 183 , 6384–6393. pmid:11591683 LaunchUrlAbstract/FREE Full Text ↵ Nag, D. K., Huang, H. V. & Berg, D. E. (1988) Gene 64 , 135–145. pmid:2840345 LaunchUrlCrossRefPubMed ↵ Wanner, B. L. (1986) J. Mol. Biol. 191 , 39–58. pmid:3540312 LaunchUrlCrossRefPubMed ↵ Metcalf, W. W., Steed, P. M. & Wanner, B. L. (1990) J. Bacteriol. 172 , 3191–3200. pmid:2160940 LaunchUrlAbstract/FREE Full Text ↵ Ausebel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1992) Recent Protocols in Molecular Biology (Wiley, New York). ↵ Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) J. Mol. Biol. 215 , 403–410. pmid:2231712 LaunchUrlCrossRefPubMed ↵ Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, O. A. (1979) Anal. Biochem. 100 , 95–97. pmid:161695 LaunchUrlCrossRefPubMed ↵ Seaver, L. C. & Imlay, J. A. (2001) J. Bacteriol. 183 , 7173–7181. pmid:11717276 LaunchUrlAbstract/FREE Full Text ↵ Nossal, N. G. & Heppel, L. A. (1966) J. Biol. Chem. 241 , 3055–3062. pmid:4287907 LaunchUrlAbstract/FREE Full Text ↵ Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97 , 6640–6645. pmid:10829079 LaunchUrlAbstract/FREE Full Text ↵ Laemmli, U. K. (1970) Nature 227 , 680–685. pmid:5432063 LaunchUrlCrossRefPubMed ↵ Metcalf, W. W. & Wanner, B. L. (1991) J. Bacteriol. 173 , 587–600. pmid:1846145 LaunchUrlAbstract/FREE Full Text ↵ Coleman, J. E. (1992) Annu. Rev. Biophys. Biomol. Struct. 21 , 441–483. pmid:1525473 LaunchUrlCrossRefPubMed ↵ Wanner, B. L. (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology (Am. Soc. Microbiol., Washington, DC). ↵ Bardwell, J. C., McGovern, K. & Beckwith, J. (1991) Cell 67 , 581–589. pmid:1934062 LaunchUrlCrossRefPubMed ↵ Pogliano, J., Lynch, A. S., Belin, D., Lin, E. C. & Beckwith, J. (1997) Genes Dev. 11 , 1169–1182. pmid:9159398 LaunchUrlAbstract/FREE Full Text ↵ Yem, D. W. & Wu, H. C. (1978) J. Bacteriol. 133 , 1419–1426. pmid:417067 LaunchUrlAbstract/FREE Full Text ↵ Jacobi, A., Rossmann, R. & Bock, A. (1992) Arch. Microbiol. 158 , 444–451. pmid:1482271 LaunchUrlPubMed ↵ Hulett, F. M., Kim, E. E., Bookstein, C., Kapp, N. V., Edwards, C. W. & Wyckoff, H. W. (1991) J. Biol. Chem. 266 , 1077–1084. pmid:1898729 LaunchUrlAbstract/FREE Full Text ↵ Bard, A. J., Parsons, R. & Joran, J. (1985) Standard Potentials in Aqueous Solutions (Dekker, New York). ↵ O'Brien, P. J. & Herschlag, D. (2001) Biochemistry 40 , 5691–5699. pmid:11341834 LaunchUrlCrossRefPubMed ↵ Snyder, S. L. & Wilson, I. B. (1972) Biochemistry 11 , 3220–3223. pmid:4558705 LaunchUrlCrossRefPubMed ↵ Neumann, H. (1968) J. Biol. Chem. 243 , 4671–4676. pmid:4879088 LaunchUrlAbstract/FREE Full Text ↵ Chlebowski, J. F. & Coleman, J. E. (1974) J. Biol. Chem. 249 , 7192–7202. pmid:4612034 LaunchUrlAbstract/FREE Full Text ↵ Reid, T. W. & Wilson, I. B. (1971) Enzymes 4 , 373–415. LaunchUrlCrossRef ↵ Albracht, S. P. (1994) Biochim. Biophys. Acta 1188 , 167–204. pmid:7803444 LaunchUrlCrossRefPubMed ↵ Lyon, E. J., Shima, S., Buurman, G., Chowdhuri, S., Batschauer, A., Steinbach, K. K. & Thauer, R. (2004) Eur. J. Biochem. 271 , 195–204. pmid:14686932 LaunchUrlPubMed ↵ Adams, F. & Conrad, J. P. (1953) Soil Sci. 75 , 361–371. LaunchUrlCrossRef ↵ Heppel, L. A., Harkness D. R. & Hilmoe R. J. (1962) J. Biol. Chem. 237 , 841–846. pmid:13906598 LaunchUrlFREE Full Text
Like (0) or Share (0)