From the Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467
Received for publication, October 9, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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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 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.
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 (
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).
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.
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 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
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.
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).
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 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
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.
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
= 18450 M
1 cm
1) due to production of
pNP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Kinetic parameters of scPLD toward various soluble and aggregate
substrates
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+.
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
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
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
max = 570 and
570 = 1800 M
1 cm
1.
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Fig. 2.
Dependence of PLD activity on iron content
(measured by ICP-MS).
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[in a new window]
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).
-helix content when this mutant was compared with scPLD. A
similar enhancement of
-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.
Metal content and relative activities of mutant PLDs
View larger version (18K):
[in a new window]
Fig. 3.
Visible absorption spectra of scPLD
and inactive mutant enzymes. scPLD ( ); C123A
(-··-); D151A (- - - -); Y154F
(- - -); H391A (····).
View larger version (13K):
[in a new window]
Fig. 5.
CD spectra of wild type scPLD ( ) and of
mutants D151A (· · ·) and Y154F (- -). H391A (not
shown) CD spectra has
-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).
View larger version (17K):
[in a new window]
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
max between 530 and 560 nm). The red shift we observe
with scPLD (
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.
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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.
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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.
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.
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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.
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REFERENCES |
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