An Iron-dependent Bacterial Phospholipase D Reminiscent of Purple Acid Phosphatases*

Carlo Zambonelli and Mary F. RobertsDagger

From the Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467

Received for publication, October 9, 2002, and in revised form, November 12, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant phospholipase D (PLD) from Streptomyces chromofuscus (scPLD) has been characterized using colorimetric assays, spectroscopic investigations, and site-directed mutagenesis. scPLD, which shows phosphodiesterase activity toward a wide variety of phospholipids and phosphatase activity toward p-nitrophenyl phosphate, exhibits a visible absorption band with lambda max at 570 nm. Metal ion analysis performed by inductively coupled plasma mass spectroscopy shows the presence of ~1 equivalent of iron, 0.27 equivalent of manganese, and 0.1 equivalent of zinc per mole of protein as isolated. The metal ion content coupled with the visible absorption feature is compatible with the presence of Fe3+-tyrosinate coordination. When scPLD was dialyzed against solutions containing Mn2+, Zn2+ or EDTA, the Fe3+ content was reduced to variable extents, and the residual specific activity correlated well with the residual iron content. Sequence homology with metal ion binding motifs in known alkaline phosphatases and purple acid phosphatase from red kidney bean shows that most of the residues involved in metal ion coordination are conserved among all the sequences considered. Mutation of some of these conserved residues (C123A, D151A, Y154F, and H391A) produced enzymes lacking iron with dramatically reduced PLD activity but little change in secondary structure or ability to bind to small unilamellar vesicles of phosphatidylcholine (with Ba2+) or phosphatidic acid. We suggest that scPLD is a member of a family of phosphodiesterase/phosphatases with structural and mechanistic similarity to iron-dependent purple acid phosphatases.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Phospholipase D (PLD,1 EC 3.1.4.4) enzymes catalyze the hydrolysis of phospholipids to phosphatidic acid and the corresponding free base. PLD activities are present in all organisms from bacteria to mammals. In higher organisms, PLDs, associated with membranes and in the cytosol, play an important role in signal transduction by production of the second messenger PA and its derivative lyso-phosphatidic acid (1). In the presence of a primary alcohol, PLD catalyzes a transphosphatidylation reaction (2-4), which has often been used to monitor PLD activity in vivo (5). Eukaryotic enzymes are activated by phosphatidylinositol 4,5-biphosphate, phosphatidylinositol 3 ,4 ,5-trisphosphate, ARF1, RhoA, and RalA (4). Cabbage PLD was also shown to be activated by the product PA (6). Most PLDs are part of a superfamily that also includes endonucleases, helicases, and lipid synthases. Members of the PLD superfamily are characterized by the presence of a conserved domain containing in its minimal element the sequence HXK(X)4D, or the HKD motif. The HKD motif has been proposed to be important for catalysis (7, 8). The PLD superfamily is classified in eight subgroups (9) based on catalytic activity, the number of HKD motifs present in the monomer, and on the requirement for Ca2+. The current model for PLD action requires that two HKD motifs join to form the active site (8, 10-12). Most of the PLD enzymes described to date contain either two HKD motifs or only one copy of this sequence, and dimerization of two identical monomers is required to form the active site (13). The involvement of two HKD motifs in the formation of the active site was confirmed by the first PLD crystal structure (12). The only known examples of PLDs not containing an HKD motif are GPI-PLD (14), phosphatidylethanol-specific PLD (PE-PLD) (15, 16) and autotaxin (17, 18), a known ectonucleotide phosphatase/phosphodiesterase recently characterized as a lysophospholipase D (19). In Ca2+-dependent PLDs, no activity is detected in the absence of this metal ion, which is believed to be an essential cofactor in substrate binding and hydrolysis (20). It should be noted that binding to vesicles or membranes and hydrolysis of substrate are most likely two events spatially separated as it was recently shown (21).

PLD from Streptomyces chromofuscus (scPLD) was previously described to be a Ca2+-dependent (22, 23) enzyme able to catalyze both hydrolysis of PC and other lipid substrates and to carry out a transphosphatidylation reaction in the presence of high concentrations of primary alcohols (see Table I) (24). Despite the observation that the highest activity is observed when the lipids are in an aggregated form (e.g. PC micelles or small unilamellar vesicles), scPLD is also able to hydrolyze a short-chain monomeric lipid such as diC4PC (see Table I). scPLD is a soluble enzyme with a molecular mass of 55 kDa that is secreted in the extracellular media where it is proteolytically cleaved to form an activated complex of 37 and 18 kDa peptides (24). When added to the extracellular media of a variety of mammalian cells, scPLD generation of PA can trigger a variety of responses (25, 26). While eukaryotic PLDs are often activated by phosphatidylinositol 4,5-biphosphate, scPLD is activated by the product PA in the presence of Ca2+ (6) and, to a lesser extent, by phosphatidylinositol phosphate (6). The details for this activation mechanism are not known.

The gene encoding S. chromofuscus PLD has been cloned (27, 28), and analysis of the primary sequence deduced from the gene sequence shows no classical HKD motif. scPLD has a requirement for mM Ca2+ for the binding and hydrolysis of PC or PE substrate presented in small unilamellar vesicles (SUVs) or in detergent-mixed micelles (6). Ca2+ is also required for optimal hydrolysis of diC4PC and diC6PC; addition of EDTA or EGTA abolishes PLD activity toward monomeric phospholipid substrate (6, 24, 28). A kinetic Kd for Ca2+ of about 60 µM was determined using soluble short-chain phospholipids as substrate (24). If no metal ion is present, scPLD binds weakly to PC vesicles. When Ba2+ is added to PC SUVs instead of Ca2+, binding of scPLD to SUVs is observed but not PC hydrolysis (23). Thus, Ca2+ seems to be an essential cofactor for the binding and hydrolysis of substrate vesicles.

In the process of characterizing recombinant scPLD, we observed a visible absorption feature consistent with the presence of a transition metal (29-31). This prompted us to: i) run a BLAST search specifically aimed at locating potential residues involved in metal ion coordination, ii) analyze metal ion content of recombinant wild type protein, and iii) prepare and characterize seven mutants (C123A, D151A, Y154F, H212A, E213A, D389A, and H391A) based on metal ion binding motifs in other phosphatases as detected with the BLAST search. That four of these mutants were inactive and had dramatically reduced iron suggests metal ion content, and iron, in particular, is critical for the activity of this unique PLD.

Continuous colorimetric assays using pNPPC and pNPP were used to further investigate the catalytic mechanism of S. chromofuscus PLD and, in particular, the apparent Ca2+ requirement of the enzyme and the ability to hydrolyze phosphodiester substrates. From the results of these studies, we propose a novel mechanism for the scPLD-catalyzed hydrolysis of phospholipids involving a binuclear iron/manganese (and possibly zinc) site as cofactor. In our model, Ca2+ plays a secondary role and is not required for catalysis. Rather it is critical for PLD to bind to phospholipid aggregates. Based on catalytic properties and sequence homology we suggest that this enzyme is a member of a novel enzyme class of phosphodiesterases related to iron-dependent alkaline phosphatases and purple acid phosphatases.

    MATERIALS AND METHODS
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Chemicals-- pNPPC, pNPP, and AP were obtained from Sigma. All the other chemicals were of the highest purity available.

PLD Purification-- The gene encoding PLD from S. chromofuscus was cloned in plasmid pTYB11 (IMPACT system, New England Biolabs) and expressed in Escherichia coli. The resulting intein-PLD fusion protein was purified by affinity chromatography on a chitin column. PLD was cleaved from the intein by incubation for 12 h with a buffer containing 50 mM DTT and eluted with the same buffer lacking DTT. Immediately after elution of PLD, the enzyme was quickly concentrated, and the DTT was removed by extensive dialysis against a buffer containing 20 mM Tris HCl, pH 8. If instead the protein was left in the high DTT solution for over 24 h, the enzyme lost a considerable amount of activity; the longer the incubation the more activity that was lost. The protein obtained after dialysis was at least 90% pure (28). Samples of scPLD were also dialyzed against buffers containing metal ions or EDTA. Excess metal ion and EDTA were removed by extensively dialyzing the samples against buffer containing 20 mM Tris, pH 8. Mutants (D151A, Y154F, H212A, E213A, D389A, H391A) were obtained by site-directed mutagenesis using the QuikChange XL kit (32). Preparation of C123A was described previously (28); this protein was supplied by Hongying Yang, Boston College. The following primers and their complements were used (the modified nucleotides are underlined): 5'-CCTGGCTGCACCTCGGCGCGTACATCTACGAGTACGGCGCC-3' (D151A); 5'-GGCGACCTGGACGCCTGGCTGCACCTCGGCGACTACATCTTCGAGTACGGCGCCGGCGAG-3' (Y154F); 5'-GGTCGTGGCGATCTGGGACGACGCGGAGATAGCCAACGACACC-3' (H212A); 5'-GCGATCTGGGACGACCACGCGATAGCCAACGACACCTGG-3' (E213A); 5'-CACGGTCTTCCTCACCGGCGCGATCCACATGGCCTGGGCC-3' (D389A); 5'-CCTCACCGGCGACATCGCGATGGCCTGGGCCAACGACGTCCC-3' (H391A).

Colorimetric Assay for PLD Activity-- PLD activity was measured as the hydrolysis of pNPPC to choline and pNPP, which in turn was further hydrolyzed by AP to pNP and inorganic phosphate. Rates, determined at least in duplicate, were calculated as absorbance at 405 nm (epsilon  = 18450 M-1 cm-1) due to production of pNP.

In other experiments, pNPP was used as a substrate and no AP was added. In both cases the kinetics were carried out in a buffer containing 20 mM Tris HCl, pH 8, 250 mM KCl, and 30 mM CaCl2. When required, CaCl2 was omitted. No EDTA was present in the assay mixtures.

UV/Visible Spectroscopy-- PLD absorption spectra were obtained on a Beckman DU-600 spectrophotometer. All the wavelength scans were obtained in the range 260-800 nm, and the absorbance at 280 was used to normalize the spectra for protein concentration.

Inductively Coupled Plasma Mass Spectroscopy-- The metal ion content of PLD samples and buffers was determined using a PerkinElmer Life Sciences ICP-MS (model PE ELAN 6100 DRC). Initially, metal ion content was determined with two methods. In the first, a minimum of 50 µg of PLD were diluted in 5 ml of 0.5% (v/v) HNO3 to digest the protein and release the metal ions. In the second method, the enzyme was ashed with a low temperature asher LFE model LTA-504 (power, 150 W; oxygen flow, 20 ml/min; vacuum, 0.46 mm Hg). The ashed material was dissolved in 0.1% HNO3. With both protocols, the solutions obtained were used without any further manipulation. Direct dilution or ashing of protein gave identical results, and we chose to use the direct dilution protocol for most of the experiments.

Matrix-assisted Laser Desorption Ionization-Time of Flight Spectroscopy-- The mass of scPLD with variable metal ion content was determined by MALDI-TOF using sinapinic acid as the matrix. PLD concentration was 20 µM in 20 mM Tris buffer, pH 8.0.

CD Spectroscopy-- Relative secondary structure elements of wild type and mutant PLD were extracted from CD spectra using an AVIV 202 spectrophotometer. Protein samples were dialyzed against 20 mM Tris buffer, pH 7.4, and diluted to a final concentration of 0.2-0.3 mg/ml. Spectra were acquired in a cuvette with optical pathlength of 0.1 cm. Each wavelength scan was acquired in the range 195-260 nm at 25 °C and deconvoluted with the program CDNN (33) using molar ellipticity between 200 and 260 nm.

Tm Determination-- The thermal denaturation (or melting) temperature of wild type and recombinant PLDs was also determined by CD spectroscopy. The temperature of the sample was varied from 25 °C to 85 °C (1 °C increments, 60 s equilibration time), and the increase in ellipticity at 222 nm was monitored. Tm values were obtained as the peak of the first derivative of each melting curve.

Vesicle Binding Assay-- PLD solutions (10 µg/ml, 500 µl total) were incubated with or without POPC or POPA SUVs at a total lipid concentration of 100 µM. Samples containing POPC also contained 1 mM EDTA or 1 mM BaCl2. The solutions were centrifuged through an Amicon microcon-100 filter to separate free from vesicle-bound enzyme. The filtrate was lyophilized and analyzed by SDS-PAGE as described previously (23).

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Substrate Specificity-- scPLD is capable of hydrolyzing phosphatidylcholine presented as monomers, micelles, or in vesicles. The product PA enhances PC cleavage in vesicles (Table I and Ref. 6). The large increase in activity seen when monomeric PC aggregates to form micelles (interfacial activation) is not observed with this phospholipase (6), although there is an enhanced activity as the acyl chain length is increased from four to six carbons (Table I). The enzyme can also catalyze the hydrolysis of PS and PE monomers but shows preference in terms of kcat/Km for PC. The monomeric compound pNPPC is also a substrate for scPLD, although kcat/Km is much lower than for any of the phospholipids substrates with a major loss in efficiency due to a weaker Km (Table I). scPLD can also carry out a transphosphatidylation reaction with primary alcohols. However, unlike HKD PLDs, a much higher amount of alcohol is necessary for the reaction. When using methanol as the alcohol, virtually no phosphatidylmethanol is detected with less than 10% methanol; in the presence of 50% of methanol, production of phosphatidylmethanol is greater than the hydrolysis product PA.


                              
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Table I
Kinetic parameters of scPLD toward various soluble and aggregate substrates

Despite the fact that scPLD catalyzes a phospholipase D reaction (hydrolysis of PC to PA and choline), it is also able to carry out a phosphatase reaction with pNPP to give pNP and inorganic phosphate (Table I). This phosphatase activity is never observed when using soluble lipids (e.g. the diC4PA from diC4PC cleavage is not hydrolyzed to diacylglycerol and inorganic phosphate) or small unilamellar vesicles (no inorganic phosphate is produced from incubating PA vesicles with scPLD) as substrates. When both pNPPC and pNPP are used as substrates, the efficiency of catalysis is significantly lower than that observed for physiological phospholipid substrates (Table I). This broader specificity is one characteristic that differentiates scPLD from the PLD enzymes in the HKD superfamily.

Ca2+-independent PLD Activity-- When PLD activity is determined using pNPPC in the presence of large enzyme concentration (>10 µg/ml), production of pNP in the coupled assay is observed in the absence of Ca2+. The activity detected under these conditions, determined as slope of the initial phase of hydrolysis (5-10 s after starting acquisition), is about 40 times lower (kcat = 0.05 s-1) than the activity measured in the presence of Ca2+. The total absence of Ca2+ from the assays where this metal ion was not added has been confirmed by ICP-MS. Neither enzyme solution or assay mixture contained Ca2+. The residual Ca2+-independent activity was abolished by the addition of 1 mM EDTA. When PLD was dialyzed against 1 mM EDTA, followed by dialysis against buffer not containing EDTA to remove the chelating agent, a partially active enzyme (~27% of the original activity) was obtained under normal assay conditions with an excess Ca2+ added (Table II). Whatever ion the EDTA is removing, it appears not to be Ca2+.


                              
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Table II
Metal content and relative activity of scPLD after dialysis with solutions containing 100 µM FeCl2, MnCl2, ZnCl2, or 100 µM EDTA and removal of excess metal ions or chelating agent

Metal Ion Content-- The EDTA inhibition of the Ca2+-independent PLD activity and the loss of Ca2+-stimulated activity upon EDTA dialysis suggests that other metal ions are involved in the mechanism of catalysis. Two distinct observations are consistent with metal ions tightly bound to PLD. (i) Prolonged incubation with DTT causes a reduction in the PLD mass of 50-60 Da, as determined by MALDI-TOF MS and <10% residual activity. (ii) scPLD shows a distinctive absorbance band in the visible region with lambda max ~570 nm (Fig. 1). This visible absorption feature is reduced when the enzyme is incubated with DTT for over 24 h. An absorbance band with this lambda max is unusual in biomolecules and is most similar to the characteristic visible absorption spectrum detected for the tyrosinate-Fe3+ complex observed in purple acid phosphatases (PAP) and some alkaline phosphatases (AP). Both these two classes of enzymes are characterized by the presence of a dimetal ion center in which one of the metals is always Fe3+ coordinated to a tyrosine hydroxyl group, while the second ion can be Fe2+, Zn2+ or Mn2+ (34). According to the generally accepted model of action for PAP, the dimetal ion center is involved in substrate binding and catalysis. ICP-MS analysis of scPLD samples confirmed the presence of 1.1 equivalent of iron and 0.27 equivalent of manganese bound to the enzyme as isolated (Table II). About 0.1 equivalent of zinc was also detected, which opens up the question as to whether Zn2+ plays a role in scPLD activity or is present as an adventitious metal ion. Other metals including cobalt, calcium, and nickel were not detected in the enzyme samples.


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Fig. 1.   Visible absorption spectra of scPLD before (------) and after dialysis of the enzyme (0.5 mg/ml) against buffers containing (from top to bottom) Fe2+ (- - -), Mn2+ (· · ·), Zn2+ (-·-·-), or EDTA (-··-··-). The characteristic absorption before dialysis with metal ion or chelating agent has lambda max = 570 and epsilon 570 = 1800 M-1 cm-1.

Dialysis of PLD samples against solutions containing 100 µM of Fe2+, Mn2+, or Zn2+ produced protein with altered metal content (Table II), spectroscopic characteristics (Fig. 1), and activity (Table II). The sample dialyzed against Mn2+ produced an enzyme with 89% activity of the isolated scPLD, visible absorption spectrum similar to that determined before dialysis, the same Mn2+ content within error, and reduced iron. When Zn2+ was present in the dialysis buffer, Zn2+ increased significantly and enzyme activity was comparable to that before dialysis. When the sample was dialyzed against Fe2+, a large excess of this iron was bound to the protein, probably due to nonspecific binding of Fe3+ ions (produced by dissolved oxygen in the buffer). Both zinc and manganese contents were reduced in this sample. The sample dialyzed against excess Fe2+ also exhibited a reduced catalytic activity (77% compared with 89 and 90% for the Mn2+- and Zn2+-dialyzed protein, respectively). Both the Zn2+- and Fe2+-dialyzed scPLD showed reduced intensity of the visible absorption band. Interestingly, residual PLD activity correlated very well with the iron content of the protein (Fig. 2) with the maximum activity at scPLD:iron = 1:1.


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Fig. 2.   Dependence of PLD activity on iron content (measured by ICP-MS).

A BLAST search with scPLD as the query does not detect any significant homology to known enzymes (most significantly other sequenced PLDs, the PLD superfamily, and non-HKD PLDs including GPI-PLD, PE-PLD, and autotaxin). With the knowledge that metal ions are bound to the protein and that they play an important role in scPLD activity, we performed a BLAST search (35) focusing on the detection of amino acid residues likely to be functionally important in metal ion binding (Fig. 4). The conserved motif in PAPs is D(X)nGD(X)nGNH(E/D)(X)nGHXH. This search identified several alkaline phosphatases with a low level of amino acid conservation (13.5%) with scPLD but with metal ion binding amino acids conserved among all sequences. Notably, Gly-150, Asp-151, and Tyr-154 are consistent with a metal ion binding site as described in kbPAP (36, 37) and other plant PAPs (38). Other residues potentially involved in metal ion coordination, though not part of a completely conserved metal ion binding site, include Gly-15, Asp-16, Cys-123, Asp-151, Tyr-154, Asp-210, Asp-211, His-212, Glu-213, Asp-389, and His-391. Positive residues highly conserved and probably involved in substrate (phosphate) binding include Arg-184, Arg-186, Arg-237, Arg-261, and Arg-368. When the alignment was extended to a member of the PAP family, the overall identity among the sequences was even lower (4.5%), but many of the residues involved in the binding of metal ions (marked by asterisks and pound symbols) were conserved (Fig. 4).


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Fig. 4.   Sequence alignment between scPLD (schPLD in the figure) and the most similar alkaline phosphatases as determined by a BLAST search using scPLD as the query sequence. Probable secreted alkaline phosphatase, Streptomyces coelicolor (scoAP1); phosphodiesterase/alkaline phosphatase D, Corynebacterium glutamicum (cglAPD); phosphodiesterase/alkaline phosphatase D, Nostoc sp. PCC 7120 (nosAPD); probable alkaline phosphatase, Streptomyces coelicolor (scoAP2); alkaline phosphatase D, Caulobacter crescentus CB15 (ccrAPD). The last sequence in the alignment (in gray) is a purple acid phosphatase from red kidney bean (rkbPAP), which was not detected by the BLAST search but which shows a similar pattern of conserved residues (common to most PAPs with only minor variations). The sequences with a black background correspond to five conserved regions in all PAPs that are involved in metal ion binding. The asterisks and pound symbols identify the residues involved in Fe3+ and Zn2+/Mn2+/Fe2+ binding, respectively, as determined from the crystal structure of PAP from red kidney bean (36).

Analysis of Mutants-- To investigate the role of single amino acid residues in the binding of metal ions, we produced the following mutants: C123A (28), D151A, Y154F, H212A, E213A, D389A, and H391A. The mutants were analyzed for their ability to catalyze the hydrolysis of pNPPC, the presence of the visible absorption band at 570 nm, and content of metal ions. Of the modified proteins C123A, D151A, Y154F, and H391A showed an almost complete loss of activity (Table III), absence of the visible absorption band (Fig. 3), and a strongly reduced content of iron (Table III). These are residues that would be predicted to be Fe3+ ligands according to the PAP alignment. The modifications H212A, E213A, and D389A had little effect on metal content, visible absorption spectrum, and on activity (data not shown). These residues aligned with PAP residues involved in chelating the divalent metal ion. We confirmed that with one exception, all the recombinant enzymes were correctly folded using CD spectroscopy to estimate secondary structure content (Fig. 5, only wild type scPLD and some of the inactive, iron-depleted mutants are shown) as well as to determine Tm values (all mutants had a Tm of 71 ± 1 °C, the same as determined for scPLD). Of the mutants considered, only H391A showed a modification in the shape of the CD spectrum. Deconvolution indicated a nearly doubled alpha -helix content when this mutant was compared with scPLD. A similar enhancement of alpha -helix content was observed for two other histidine mutants of this enzyme (28). All proteins also had similar binding properties to PC and PA vesicles (Ba2+ needed for the enzymes to partition to PC vesicles but tight binding exhibited to PA vesicles with EDTA present) indicating that phospholipid binding sites (at least an interfacial binding site) were intact (Fig. 6). The ability of all these PLD enzymes to bind to substrate PC SUVs in a Ba2+-dependent fashion, characteristic of wild type scPLD, was not affected in any of the mutations.


                              
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Table III
Metal content and relative activities of mutant PLDs


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Fig. 3.   Visible absorption spectra of scPLD and inactive mutant enzymes. scPLD (------); C123A (-··-); D151A (- - - -); Y154F (- - -); H391A (····).


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Fig. 5.   CD spectra of wild type scPLD (------) and of mutants D151A (· · ·) and Y154F (- -). H391A (not shown) CD spectra has alpha -helix content doubled if compared with wild type scPLD. The spectra were acquired at 0.5-nm intervals at 25 °C. Deconvolution was performed with the program CDNN version 2 (33).


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Fig. 6.   SDS-PAGE showing Ba2+-dependent binding of native and mutant PLDs to POPC substrate vesicles. The assay was carried out as described previously (23).


    DISCUSSION
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ABSTRACT
INTRODUCTION
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The HKD domain is the fingerprint of a large class of PLD enzymes. These members of the PLD superfamily contain one or two HKD sequences that are believed to play a key role in substrate binding and removal of the polar head of phospholipids by catalytic cleavage of the distal phosphoester bond. This reaction should proceed through release of the base (choline in the case of PC substrate) and concomitant formation of a covalent intermediate in which the phosphate group belonging to the phospholipid is bound to the histidine from one of the HKD domains. Subsequent hydrolysis of the covalent intermediate releases phosphatidic acid from the PLD. In this mechanism, the His residue belonging to the other HKD domain acts as a general acid.

scPLD is functionally a phospholipase D since it clearly carries out both phospholipid hydrolysis and transphosphatidylation (Table I). Nonetheless, its primary sequence as determined by translation of the cloned gene does not contain an HKD domain. Furthermore, this protein contains two metal ions, iron and manganese or possibly zinc, when isolated. The protein:metal ion molar ratios we determined (PLD:iron:manganese:zinc = 1:1:0.27:0.11) are reminiscent of the metal ion content of many purple acid phosphatases when isolated and not supplemented with divalent ions (36-39). In PAPs the dimetal center is formed by Fe3+ and Fe2+, Zn2+, or Mn2+. Fe3+ is coordinated to the hydroxyl group of a Tyr residue, and this complex is responsible for the characteristic purple color of the protein (typical lambda max between 530 and 560 nm). The red shift we observe with scPLD (lambda max ~570 nm) could be consistent with the coordination of Fe3+ by the residues Cys, Tyr, Asp, and His instead of Asp, Tyr, Asp, and His. The Cys residue in scPLD is not accessible to chemical modification by DTNB (5,5'-dithiobis(nitrobenzoic acid)) or related reagents,2 although this could also be explained if the Cys is buried in the protein interior.

The absence of the characteristic HKD domain, the likely presence of a dimetal center, and the analogy in putative metal ion binding residues with PAP and AP suggest that the mechanism of catalysis of PLD from S. chromofuscus is substantially different from the model currently proposed for other PLD-catalyzed phospholipid hydrolysis. We propose that correct substrate binding and hydrolysis occurs through a mechanism similar to the one described for PAP (34, 39). The phosphate group of PC, through two of its oxygens, acts as a bidentate ligand coordinated to both metal ions; this increases the electrophilicity of the neighboring phosphorus atom. Substrate binding is likely further stabilized by adjacent Arg or Lys residue(s). Fe3+ also acts as a general acid by binding a water molecule and generating a metal-bound hydroxyl group at physiological pH and positioning it in an optimal orientation for substrate hydrolysis. If a primary alcohol is bound instead of a water molecule, a transphosphatidylation reaction can occur. In contrast to transphosphatidylation by HKD-PLD enzymes, the scPLD reaction would likely require higher alcohol concentrations to displace water from the Fe3+ site (and this has in fact been observed). The metal ion(s) would also participate in the stabilization of the partially negatively charged transition state.

To test the hypothesis that scPLD binds metal ions in a fashion similar to PAP, we produced mutants for residues we identified as putative metal ligands by sequence alignment with published PAPs and APs. The alignment showed a very low overall level of residue conservation. However, there was almost complete conservation of metal binding residues when aligning scPLD with kbPAP. A similar case of very low identity but complete conservation of metal ion binding residues (with the only exception of Tyr, which is substituted by a solvent molecule) (40) was previously described when comparing purple acid phosphatases and protein phosphatases (41). Recently, a similar approach based on a data base search and detection of a small number of highly conserved functionally relevant residues allowed the identification of a novel phytase as a homologue to purple acid phosphatase (42). A careful inspection of the alignment we produced allowed us to identify four residues that once mutated (C123A, D151A, Y154F, and H391A) generated proteins that were characterized by lower iron content, no visible absorption band, and almost completely abolished PLD activity. Interestingly, comparing the sequences of scPLD and kbPAP (Fig. 4) and considering the latter four residues, three (Asp-151, Tyr-154, and His391) correspond to residues predicted to be involved in Fe3+ binding and one (Cys-123) aligns with Asp-135, which is a ligand of Fe3+ in kbPAP. We also obtained mutants of residues suggested by sequence alignment to be involved in metal ion, but not iron, binding based on the alignment with kbPAP (H212A, E213A, and D389A). These mutants showed unaltered iron content and activity similar to scPLD. Vesicle binding behavior, CD spectra, and Tm values were indiscernible from those determined for scPLD and the inactive recombinant proteins we obtained. This suggests that if scPLD has a dimetal ion site that is critical for catalysis, it is not exactly the same as for the PAP and related proteins since modification of putative ligands of the divalent ion do not affect enzyme activity or visible absorption spectrum.

Other examples of non-HKD PLDs are known. Recent reports showed that autotaxin, an enzyme known to possess 5'-nucleotide pyrophosphatase and 5'-nucleotide phosphodiesterase activities (43), is a lysophospholipase D that does not contain an HKD motif (18, 19). However, it shows striking dissimilarities when compared with PAP, AP, or scPLD. None of the amino acids involved in metal binding in PAP and AP are conserved when aligning these sequences with autotaxin (data not shown). Although autotaxin was shown to be activated by the addition of Co2+ (19), its activity does not appear to be absolutely dependent on Fe3+ or any other metal ion. Furthermore, autotaxin is active also in the absence of Co2+. When physiologically relevant phospholipids are used as a substrate, addition of Co2+ induces less than a 2-fold activation. Other reported examples of non-HKD PLD enzymes include GPI-PLD (14) and PE-PLD (15, 16). While the former requires metal ions for activity (Ca2+ and Zn2+), none of these enzymes have been reported to require iron. Although the protein sequence of PE-PLD is not yet known, the GPI-PLD primary sequence does not contain any element suggesting the presence of possible metal ion binding clusters similar to the ones detected in PAPs and now in scPLD.

Until recently, the observation that PLD activity was completely inhibited by EDTA or EGTA addition led to the conclusion that Ca2+ was an essential cofactor for PLD. In this study we demonstrate that Ca2+ is a nonessential activator of PLD from S. chromofuscus. Ca2+ may be involved in correct or more efficient substrate binding as previously proposed (44). An alternative role for Ca2+ could be to stabilize and enhance the release of the product PA by neutralizing the partial negative charge in the intermediate state in a similar way as that described for Fe3+ in the case of PAP (34, 39). Thus, when pNPPC and pNPP are used as substrates, scPLD can be defined as a phosphodiesterase/phosphatase with phosphatase activity similar to PAP. Several possible functions have been proposed for PAPs of different origin, but the physiological substrate(s) of these enzymes has yet to be identified. scPLD appears to be the first member of a new family of Ca2+-activated, iron/manganese-dependent phospholipase D/phosphatases. Perhaps PAP (and some APs) are members of the same family and thus may be involved in phospholipid degradation.

    ACKNOWLEDGEMENT

We thank Dr. Gordon Wallace and Franco Pala of the Environmental, Coastal, and Ocean Sciences Department at the University of Massachusetts, Boston, MA, for helping us obtain the ICP-MS results.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 26762 (to M. F. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Merkert Chemistry Center, Boston College, 2609 Beacon St., Chestnut Hill, MA 02467. Tel.: 617-552-3616; Fax: 617-552-2705; E-mail: mary.roberts@bc.edu.

Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M210363200

2 C. Zambonelli, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; PA, phosphatidic acid; AP, alkaline phosphatase; diC4PC, dibutyroylphosphatidylcholine; diC6PC, dihexanoylphosphatidylcholine; diC6PE, dihexanoylphosphatidylethanol; GPI-PLD, glycosylphosphatidylinositol-specific PLD; PE-PLD, phosphatidylethanol-specific PLD; PAP, purple acid phosphatase; kbPAP, red kidney bean purple acid phosphatase; scPLD, S. chromofuscus phospholipase D; ICP-MS, inductively coupled plasma mass spectroscopy; pNPP, p-nitrophenylphosphate; pNPPC, p-nitrophenylphosphocholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPA, 1-palmitoyl-2-oleoyl-phosphatidic acid; SUV, small unilamellar vesicle; DTT, dithiothreitol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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