From the Department of Biological Chemistry,
University of Michigan Medical School, Ann Arbor, Michigan 48109,
Department of Biological Chemistry, University of California
School of Medicine, Davis, California 95616, and ** Pulmonary-Critical
Care Medicine Branch, NHLBI, National Institutes of Health,
Bethesda, Maryland 20892-1434
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ABSTRACT |
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A phospholipase D (PLD) superfamily was recently
identified that contains proteins of highly diverse functions with the
conserved motif
HXKX4DX6G(G/S).
The superfamily includes a bacterial nuclease, human and plant PLD
enzymes, cardiolipin synthases, phosphatidylserine synthases, and the
murine toxin from Yersinia pestis (Ymt). Ymt is
particularly effective as a prototype for family members containing two
conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of
pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to
have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol)
phosphate as a substrate. Using these substrates, the amino acids
essential for Ymt function were determined. Specifically, substitution
of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of
104 and phospholipid turnover to an undetectable level. The
role of the conserved residues in catalysis was further defined by the
isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the
catalytic reaction. Based on these data, a unifying two-step catalytic
mechanism is proposed for this diverse family of enzymes.
Human phospholipase D
(PLD)1 is an effector in
multiple signaling cascades. Activity of this lipid-modifying enzyme
is, therefore, tightly regulated by multiple activators including small
guanine nucleotide-binding proteins from the ADP-ribosylation factor
and Rho families, phosphatidylinositol 4,5-bisphosphate, and protein kinase C (reviewed in Ref. 1). Human PLD1 catalyzes the hydrolysis of
phosphatidylcholine to generate phosphatidic acid and choline. Phosphatidic acid can be further hydrolyzed to lysophosphatidic acid by
phospholipase A2 or to diacylglycerol by phosphatidic acid
phosphohydrolase. PLD can also catalyze a transphosphatidylation reaction in which an alcohol (usually ethanol) substitutes for water in
the reaction, resulting in the formation of the alcohol derivative of
phosphatidic acid (typically phosphatidylethanol); this assay is
commonly employed to measure PLD activity (2). Other lipid-modifying
enzymes such as cardiolipin and phosphatidylserine synthases also
catalyze transphosphatidylation reactions.
PLD is one of a group of enzymes with diverse functions that has been
referred to as the PLD superfamily (3-5). All members contain a
conserved motif,
HXKX4DX6G(G/S).
Members of this family that contain a single copy of the conserved
motif include a bacterial endonuclease (Nuc) and a helicase-like
protein from Escherichia coli (6-7). Most other family
members contain two copies of the conserved motif
(HXKX4DX6G(G/S)),
including human and plant PLD enzymes (3, 8), cardiolipin synthase
(9-10), phosphatidylserine synthase (11), and a murine toxin from
Yersinia pestis (12). This signature motif is not, however,
absolutely conserved, and variation exists in many of the conserved
residues in the motif (4, 13).
The Y. pestis murine
toxin (Ymt) is encoded on a 110-kilobase plasmid that is
unique to the bacterium which causes the plague (12). Y. pestis is transferred between mammalian hosts via a flea vector.
As a known determinant for plague virulence, Ymt is believed to play a
role in Y. pestis pathogenesis, both in the rodent host and
the flea vector (14-15). For example, Ymt may contribute to the more
rapid death of mice injected with Y. pestis, as compared
with mice injected with other Yersinia species (16). In
addition, Ymt is required for Y. pestis survival in the
flea.2
Ymt is a 586-amino acid protein with two conserved motifs,
HXKX4DX6G(G/S).
It is similar to human PLD in having two of the superfamily signature
motifs, but it is substantially smaller than the 1074-residue human PLD
protein. The similarities in the two conserved motifs suggested that
Ymt and PLD might utilize a common catalytic mechanism. Finally, an
understanding of the activity and mechanism of Ymt would afford a
rational approach for exploring the role of Ymt as a virulence factor
for the plague bacterium, Y. pestis.
We have, therefore, investigated the physical properties, function, and
catalytic mechanism of the cloned toxin as a prototype for superfamily
enzymes with two conserved motifs. In this report, we demonstrate that
Ymt has PLD-like activity as assessed by the hydrolysis of
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine
as well as the transphosphatidylation reaction involving
phosphatidylcholine. In addition, we have identified artificial
substrates for Ymt to explore the catalytic mechanism. These substrates
have allowed us to demonstrate that the invariant histidine and lysine
residues found in the conserved motifs of all superfamily members are
essential for catalysis. We have also shown that the catalysis by Ymt
proceeds via a phosphoenzyme intermediate. Based on our findings, we
propose a unifying, two-step mechanism for substrate hydrolysis by
dual-domain PLD superfamily members. The expression of large
concentrations of recombinant Ymt also allowed us to crystallize this
protein, setting the stage for structural analysis of this member of
the PLD superfamily.
Materials
Bis(p-nitrophenyl) phosphate (bis-pNPP) and Sephadex
G-100 were purchased from Sigma. Immobilon P membrane was obtained from Millipore. Nickel nitrilotriacetic acid-agarose was purchased from Qiagen. 32P-Labeled inorganic phosphate (9000 Ci/mmol),
L- Construction of Expression Plasmids
Wild-type Ymt was amplified from the 110-kb plasmid from the KIM
strain of Y. pestis provided by J. Hinnebusch. The 5' primer incorporated a restriction site for NdeI
(GTCATGCATATGCTTCAAATAGATAATCTCA), and the 3' primer added
a SalI restriction site and a polyhistidine tag
(GACGTCGACTTA(GTGATG)3ATTTGGGCTTAATTTTGGAA).
The PCR product was ligated into the
NdeI/SalI sites of a modified pT7-7 expression vector, which contains the lac operator (Ref. 17, provided
by J. C. Clemens). The entire coding sequence was verified by
sequencing. Using the wild-type Ymt construct as a template, the
following mutations were made using the TransformerTM site-directed
mutagenesis kit (CLONTECH): H188N, H524N, K190S,
K526S, D195N, D531N, D195E, D531E, S539T, and S539A. For mutants with
changes in two conserved residues, primers for both substitutions were
utilized in conjunction. All mutations were verified by sequencing.
Expression and Purification of Ymt
The pT7-7 Ymt construct was used to transform Novoblue DE3
(Novagen) E. coli by standard methods. Single colonies were
used to inoculate 1 liter of 2× YT medium containing ampicillin (100 µg/ml). Cultures were grown overnight at 37 °C to an optical
density (600 nm) of 0.5-0.9, induced by the addition of isopropyl
Crystallization of Ymt
Full-length Ymt was diluted to 5 mg/ml in 10 mM
NaCl, 5 mM Tris, pH 7.4, and screened for crystallization
conditions. Crystals (0.01 × 0.02 × 0.04 mm3)
formed in condition #33 (2.0 M ammonium formate and 100 mM Hepes, pH 7.5) of the Hampton Research Crystal Screen
II. Larger crystals (0.1 × 0.1 × 0.1 mm3) of
full-length Ymt were grown by macroseeding. Crystals grown in 1.35-1.6
M ammonium formate, 1% polyethylene-200, 100 mM Hepes, pH 7.5, were placed in equilibrated 10-µl
hanging drops containing 5 µl of protein solution, 15 mg/ml, and 5 µl of 1.5 M ammonium formate containing 100 mM Hepes, pH 7.5, and allowed to grow for 21 days at
22 °C. The macroseeded crystals were frozen in 20% glycerol, 1.5 M ammonium formate, 100 mM Hepes, pH 7.5, and
diffraction data were collected on a Rigaku RU300 rotating anode
equipped with a RAXIS IV detector.
Physical Characterization of Ymt
UV spectra of wild-type and mutant enzymes were recorded over a
wavelength range of 240-310 nm at 25 °C using a Perkin-Elmer l6
spectrophotometer in assay buffer (25 mM Tris, 25 mM Bis-Tris, 50 mM acetate, pH 6.0). The final
concentration of all proteins was 0.250 mg/ml in a final volume of 0.5 ml. Circular dichroism (CD) spectra were recorded using a JASCO J-710
spectropolarimeter at 25 °C over a wavelength range of 178-260 nm.
The final concentration of each protein was 0.1 mg/ml in distilled
water. Size exclusion chromatography was used to determined the
oligomeric state of purified recombinant Ymt. Ymt (100 µg) was
injected onto a Superose 6 column (Amersham Pharmacia Biotech) using 30 mM Tris, pH 7.4, 150 mM NaCl as the mobile
phase at a flow rate of 0.5 ml/min. Protein elution was monitored by
absorbance at 280 nm.
The concentration of the purified protein was quantified by the
University of Michigan Protein Core Facility (amino acid analysis performed on a Perkin-Elmer Applied Biosystems model 420H
hydrolyzer/derivatizer and 130A Separation System). Amino terminal
sequence analysis was performed using a Perkin-Elmer Applied Biosystems
494 sequenator.
Artificial Substrate Hydrolysis
All assays were performed at 30 °C in assay buffer pH 6.0. For substrate screening, 1 µM of wild-type Ymt was
incubated with bis(para-nitrophenyl) phosphate (2-20
mM), para-nitrophenyl phenyl phosphonate (2-100
mM), thymidine 5'-monophosphate para-nitrophenyl ester (2-50 mM), para-nitrophenyl phosphate
(2-100 mM), or para-nitrophenyl phosphorylcholine (2-50 mM). Stock solutions of each
substrate were made in assay buffer, adjusted to pH 6.0, diluted, and
stored at 30 °C until use. For mutant enzyme assays, Ymt was
incubated with 20 mM bis-pNPP in assay buffer at 30 °C.
Final enzyme concentrations were as follows: 1 µM for
wild-type Ymt, 10 µM for Ymt mutants with single amino
acid substitutions, and 50 µM for Ymt with two amino acid
substitutions. Product release (para-nitrophenolate) was
monitored at 405 nm. Initial rates of enzyme-catalyzed hydrolysis were
determined from continuous measurements taken over 3 min for wild-type
Ymt or discontinuous readings taken over 2 h for Ymt mutants.
Rates were calculated using the molar extinction coefficient for pNP,
18,000 M Physiological Substrate Hydrolysis
Nuclease Assay--
The nuclease assay was performed as
described previously (19). Briefly, calf thymus DNA (7.5 µg) was
incubated with 0-3 µg of Nuc or Ymt for 20 min at 37 °C in assay
buffer with 100 µM EDTA. Samples were resolved using a
1% agarose gel, and DNA was visualized under a UV light source.
Phosphatidylcholine Hydrolysis--
Phosphatidylcholine vesicles
were prepared by mixing
L- Phospholipid Headgroup Release Assay--
All assays were
performed in 50 mM Tris, pH 7.4, 80 mM KCl at
37 °C for 1 h. Headgroup-labeled phosphatidylcholine,
phosphatidylethanolamine, and phosphatidylserine (as sonicated
vesicles; final concentration, 10 µM) were incubated with
wild-type Ymt (final concentration, 5 µM) in 150 µl for
1 h. The entire reaction was extracted in 1 ml of
chloroform:methanol:concentrated HCl (50:50:0.5) and 350 µl of 1 mM EGTA in 1 M HCl. A portion of the upper,
aqueous phase was subjected to scintillation counting.
Transphosphatidylation Assay--
To measure phosphatidic
acid formation and transphosphatidylation, sonicated
phosphatidylcholine vesicles were prepared from chain-labeled
L- Formation and Properties of the Ymt Phosphoprotein
Bovine serum albumin, wild-type Ymt, or mutant Ymt was incubated
at a final concentration of 30 µM with 1 mM
potassium [32P]phosphate (15 Ci/mmol) at 25 °C in 200 mM acetate, pH 4.5. Typically, binding effeciency was
0.04-0.05%. Over 10 min, aliquots of the reaction were removed and
quenched with an equal volume of carbonate buffer (1 M
Na2CO3, pH 11, 0.5% SDS). Sample buffer (12 mM Tris, pH 6.8, 5% glycerol, 0.4% SDS, 2.9 mM Phosphoamino Acid Analysis
Ymt was labeled as described above and subjected to SDS-PAGE
using a 10% gel. Slices of acrylamide containing labeled Ymt were
excised and submerged in 3 N KOH at 105 °C for 5 h.
The resulting hydrolysate was diluted 400-fold with water containing
internal standards. Phosphoamino acids were separated by ion-exchange
chromatography (21). O-Phthalaldehyde was added to the
eluate, and the resulting fluorescence was detected on-line (21).
Radioactivity was quantified by liquid scintillation counting.
Phospholysine and phosphohistidine were synthesized as described
previously (21). All other standards were purchased from Sigma.
Purification, Enzymatic Activity, and Crystallization--
Ymt is
a member of the PLD superfamily which, like human and plant PLD,
contains two conserved motifs,
HXKX4DX6G(G/S)
(Fig. 1A). The sequence for
Ymt previously reported by Cherepanov et al. (12) contained
three potential initiation methionine residues at positions 1, 42, and
56. We attempted to express recombinant protein using each of these
residues as initiation codons. However, recombinant proteins starting
with Met-42 and Met-56 were insoluble. Therefore, we utilized Met-1 as
the initiation methionine for the protein used in our studies. Using
this construct, Ymt containing a carboxyl-terminal hexahistidine
"affinity tag" was overexpressed and isolated using a single-step
procedure to
Ymt has no known native substrates and no well defined catalytic
activity. The PLD superfamily members include nucleases, lipid-synthesizing enzymes, and lipid-degrading enzymes. The one feature of all the substrates utilized by this diverse array of proteins is the phosphodiester bond. We reasoned that physiological substrates as well as artificial substrates would have to contain a
hydrolyzable phosphate diester. Therefore, we explored nucleic acids
and phospholipids as potential Ymt substrates. Ymt failed to hydrolyze
DNA (data not shown); however, Ymt did hydrolyze phosphatidylethanolamine and to a lesser extent,
phosphatidylcholine and phosphatidylserine in a headgroup release assay
(Fig. 2). In a separate headgroup release
assay, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate
(at 75 and 42 nM, respectively, the highest concentation
possible in the assay), only 0.2 pmol of headgroup was released from
phosphatidylinositol/h with Ymt, whereas phosphatidylinositol
4,5-bisphosphate was not cleaved. Upon TLC analysis, it was clear that
Ymt cleaved the terminal phosphodiester bond of
L-
Because end point lipid hydrolysis assays provide only a qualitative
assessment of Ymt activity, we explored artificial substrates to
facilitate quantitation of Ymt catalytic activity. Multiple phosphomonoester and phosphodiester substrates were screened as potential Ymt substrates (See "Experimental Procedures"). The best
artificial substrate tested was the phosphodiester, bis-pNPP (Fig.
3). Ymt hydrolyzed bis-pNPP to pNP and
pNPP. pNPP is not a substrate for Ymt; thus, this enzyme is a
phosphodiesterase and will not function as a phosphatase on phosphate
monoesters.
Hydrolysis of bis-pNPP by Ymt can be measured in a continuous assay and
was found to be linear over 3 min at several substrate concentrations
(Fig. 3A). When initial velocities were plotted against the
substrate concentrations, the reaction did not appear to follow
Michaelis-Menton kinetics (Fig. 3B). Limiting solubility (26 mM) of bis-pNPP prohibited determination of the kinetic
parameters for hydrolysis. The artificial substrate does, however,
afford a quantitative measure of activity and has a number of
advantages over the end point assay employing phosphatidylcholine.
The large quantities of pure Ymt allowed us to search for
crystallization conditions for this protein. Ymt formed small rod-like crystals in 2.0 M ammonium formate and 100 mM
Hepes, pH 7.5, and diffraction-quality crystals were obtained by
macroseeding (Fig. 4). The macroseeded
crystals frozen in a solution containing 20% glycerol, 1.5 M ammonium formate, and 100 mM Hepes, pH 7.5, diffracted to 3.5 Å resolution. These conditions provide the necessary
starting point for determining the structure of Ymt by x-ray
crystallography.
Mutagenesis of Amino Acids Located in the Two Conserved
Motifs--
Recently, Sung et al. (22) found that
site-directed mutagenesis of amino acids in the conserved motifs of
human PLD1 resulted in a marked loss of PLD activity. These
investigations employed human PLD produced by transiently transfected
COS-7 cells. The limited quantity of enzyme obtained from cell lysates
prohibited a detailed analysis of the role of these residues in PLD
catalysis. To define the role of the conserved residues in the two Ymt
signature motifs, histidines in the first and/or second conserved motif of Ymt were replaced with asparagine by site-directed mutagenesis (H188N, H524N, and H188N/H539N). Using similar methodology, lysines were substituted with serine (K190S, K526S, K190S/K526S), and the
aspartic acids were replaced with asparagine or glutamic acid (D195N,
D531N, D195N/D531N, or D/E). In addition, a conserved serine in the
second motif was replaced by threonine or alanine (S539T or S539A,
Table I). Although substitution of the
conserved aspartic acid in either domain rendered the enzyme insoluble, all other mutations yielded soluble proteins that were purified to
homogeneity (Fig. 5).
As the mutations may potentially disrupt secondary structure, the
structure of each mutated enzyme was evaluated using UV absorbance, CD,
and size-exclusion chromatography. The UV and CD spectra of the Ymt
mutants were identical to that of wild-type Ymt suggesting that
replacement of residues in the conserved motifs did not disrupt the
secondary structure of this enzyme (data not shown). In addition, the
oligomeric state of the Ymt was assessed using size-exclusion
chromatography. Amino acid analysis as well as the cDNA sequence
predicted a protein of 61.1 kDa which is consistent with the size
determined by SDS-PAGE (63 kDa, Fig. 1B). However, all
proteins eluted from a Superose 6 size-exclusion column at an apparent
molecular mass of 125 kDa, which suggests that wild-type and mutant Ymt
exist as dimers (data not shown).
In an effort to functionally evaluate these mutant enzymes, we employed
the physiological and artificial substrates for wild-type Ymt,
phosphatidylcholine and bis-pNPP, respectively. Replacement of both
conserved histidines or lysines reduced phosphatidylcholine hydrolysis
by Ymt to an undetectable level (data not shown). Artificial substrate
hydrolysis was also markedly reduced as a result of substitutions in
the conserved motifs (Table I). Substitution of the asparagine for
histidine in the first conserved motif (H188N) reduced the turnover
rate of bis-pNPP 164-fold. Similarly, mutagenesis of the histidine in
the second conserved motif (H524N) reduced the rate of hydrolysis
470-fold. Replacement of both conserved histidines in the signature
motifs resulted in a multiplicative functional loss and reduced the
turnover rate 16,400-fold. Mutagenesis of the lysine residues in the
conserved motifs resulted in similar losses of activity (Table I).
Based on the catalytic activity of human PLD produced in COS-7 cells,
Sung et al. (22) concluded that the conserved serine in the
second domain
(HXKX4DX6G(G/S))
served as a nucleophile in catalysis. We explored the role of this
conserved serine in Ymt catalysis using the artificial substrate,
bis-pNPP. Mutagenesis of Ser-539 in Ymt to threonine resulted in a
2-fold reduction in the rate of bis-pNPP hydrolysis, and replacement of
this amino acid with alanine reduced the rate by 12-fold. We conclude
that the histidines and lysines in the conserved motifs are critical for Ymt function and the conserved serine in the second motif serves a
nonessential role in catalysis.
Numerous examples of two-domain proteins where the domains function
independently have been described. Two examples of this class of
proteins include the receptor protein tyrosine phosphatases (PTPases),
LCA (leukocyte common antigen) and LAR (LCA-related molecule). These
receptors contain two highly homologous cytoplasmic domains that appear
to function independently (23). For example, mutagenesis of key
catalytic residues in the first cytoplasmic domain of either PTPase
abolishes more than 99% of the PTPase activity, whereas analogous
mutations in the second cytoplasmic domain had little or no effect on activity.
Unlike the functionally independent domains of the receptor protein
tyrosine phosphatases, the domains of the Ymt dimer appear to display
functional dependence. Substitution of a single conserved residue in
either domain reduced the catalytic activity by more than 99%. Several
models were developed to explain these data. Fig.
6 depicts three possible dimer
configurations for mutant Ymt with a single substitution in one
conserved histidine (H Dissection of the Mechanism of Ymt Catalysis--
There are at
least two possible mechanisms by which Ymt could accomplish substrate
turnover. Ymt catalysis could proceed via a single displacement
reaction that does not involve the formation of an enzyme intermediate
(Fig. 7A), or Ymt catalysis
could be accomplished by a two-step reaction involving the formation
and breakdown of a covalent intermediate (Fig. 7B). To
discriminate between these possibilities, we took advantage of the fact
that a number of phosphohydrolases have been shown to catalyze
phosphate (oxygen)-water exchange (24). We asked if Ymt would similarly catalyze phosphate (oxygen)-water exchange using
32P-inorganic phosphate. If the reaction pathway shown in
Fig. 7A is used by Ymt, no incorporation of label would be
seen in the enzyme. If, however, the mechanism shown in Fig.
7B is employed, then it might be possible to "trap"
32P-labeled phosphate covalently bound to the enzyme. Ymt
was incubated at pH 4.5 with 32P-labeled phosphate. Rapid
denaturation and analysis by SDS-PAGE demonstrated that
32P-labeled phosphate was covalently bound to Ymt (Fig.
8A). Substitution of the
conserved serine (S539T and S539A) in Ymt resulted in a reduction in
the amount of 32P-labeled protein. Substitution of a single
conserved histidine (H188N, Fig. 8A) or lysine (data not
shown) residue in the conserved motifs of Ymt, however, prohibited the
formation of a covalent phosphoprotein. These data argue that although
the conserved histidines and lysines in the signature motifs are
essential for phosphoenzyme formation, the conserved serine is not.
We attempted to further characterize the enzyme intermediate by
determining the amino acid in Ymt that formed the enzyme-phosphate linkage (25-26). To evaluate the stability of the phosphoenzyme, the
labeled Ymt phosphoprotein was transferred to Immobilon P membrane.
Radiolabeled Ymt bound to the membrane was stable under alkaline
conditions (1 M NaOH) but labile on exposure to acid (1 N HCl) or 1 M hydroxylamine (Fig.
8B, top panel). Membranes containing the Ymt
phosphoprotein were stained with Coomassie Blue after incubation to
verify that the protein remained bound (Fig. 8B,
bottom panel). The stability of the phosphoprotein was also
examined over a wide range of pH conditions, and the resulting hydrolysis curve paralleled that of phosphohistidine (Fig.
8C, 27, 28). Finally, we identified the labeled amino acid
directly by ion-exchange chromatography after complete alkaline
hydrolysis of the labeled intermediate (Fig.
9). The only radiolabeled amino acid
detected in the hydrolysate co-eluted with phosphohistidine, with the
remainder of the radiolabel being inorganic phosphate. Collectively,
these data suggest that Ymt catalysis proceeds via a phosphohistidine
intermediate as shown in Fig. 10. In
the proposed catalytic mechanism, the histidine in the conserved motif
HXKX4DX6G(G/S) serves as the nucleophile and forms a phosphoenzyme intermediate. Although there is no evidence to support the role of histidine from
another domain of the dimer serving as a general acid in catalysis,
this suggestion is consistent with the dimeric state of the enzyme. In
our suggested mechanism, a histidine in one domain serves as the
nucleophile to attack the phosphodiester bond, whereas a histidine from
the another domain acts as a general acid to donate a proton to the
leaving group to facilitate intermediate formation. The phosphoenzyme
intermediate is subsequently broken down by hydrolysis.
We report for the first time the physiochemical properties,
crystallization, a potential native substrate, and the mechanism of
catalysis for this dual-domain PLD superfamily enzyme. We demonstrate that for this PLD superfamily member, each signature motif
(HXKX4DX6G(G/S)) is required for catalysis. Rudge et al. recently determined
that the conserved lysine in the second motif in the yeast PLD, SPO14, is essential for PLD function in meiosis (29). Consistent with these
findings, substitution of any one of these residues in either conserved
motif of Ymt markedly reduced enzymatic activity. Furthermore, mutagenesis of both histidine or lysine residues in the Ymt active site
resulted in a multiplicative functional loss. Our findings are also
consistent with the recent observations of Xie et al. (30),
who demonstrated that co-expression of the amino- and carboxyl-terminal
domains of rat brain PLD resulted in active proteins, whereas
expression of either domain alone in COS cells produced no catalytic
activity. The conclusions from these studies by Exton and coworkers (1)
is that the two halves of PLD can associate, and that this may be
essential to bring the two HKD domains together to from an active site.
Sung et al. (22) suggested a model for PLD catalysis in
which the conserved serine (Ser-539 in Ymt) functions as the
nucleophile in catalysis. The model was based on assays of cell lysates
from PLD-transfected COS-7 cells. We propose a catalytic mechanism where the conserved histidine in one domain serves as the nucleophile, and the histidine from another domain facilitates the release of the
leaving group by protonation. The role of histidine as the nucleophile
in catalysis is supported by biochemical studies of both Ymt and the
single domain PLD superfamily member, Nuc (31). Mutation of the single
conserved histidine in Nuc or both conserved histidines in Ymt reduced
the rate of artificial substrate hydrolysis by a factor of
105. In contrast, mutagenesis of Ser-539 in Ymt reduced the
rate of bis-pNPP hydrolysis only by a factor of 10. In addition,
substitution of the conserved serines in Ymt or Nuc had little impact
on intermediate formation, whereas mutagenesis of any single conserved
histidine abolished intermediate formation. The chemical properties of
the enzyme intermediate were also consistent with histidine and not with serine. Finally, phosphoamino acid analysis identified
phosphohistidine as the only radiolabeled amino acid in the labeled
enzyme intermediate.
Our data demonstrate that Ymt catalysis proceeds via a two-step
mechanism involving the formation and breakdown of a phosphoenzyme intermediate. It is known that the single-domain family member, Nuc,
also utilizes a two-step catalytic mechanism (29). In addition, hydrolysis of substrates by cabbage PLD and E. coli
phosphatidylserine synthase (two members of the PLD superfamily)
proceeds with retention of configuration, suggesting a reaction
mechanism involving two steps: the formation and breakdown of an enzyme
intermediate (32-33).
Although the crystal structure of this dual-domain PLD superfamily
member has not been completed, the structure of the single-domain superfamily enzyme, Nuc, has been solved (34). Enzymes in the PLD
superfamily are likely to share both a common catalytic mechanism and
common structural features. The structure of Nuc provides an indication
of how conserved residues in Ymt might function. For example, in the
structure of Nuc complexed with the competitive inhibitor, tungstate,
conserved histidines from two monomers of Nuc are coordinated to the
tungstate ion. These residues are properly positioned to serve as the
nucleophile and the general acid in catalysis. The conserved lysine
residues also interact with the tungstate ion. It was concluded from
the structure that the lysine residues bind and stabilize the
negatively charged oxygen atom(s) on the phosphate diester. The
biochemical analysis of Ymt described in this study is consistent with
the structural data from Nuc. The catalytic role of the residues in the
conserved motifs is likely to be the same not only for Ymt and Nuc but
for other PLD superfamily members as well. Although the proteins in the
superfamily have diverse physiological functions and substrates that
range from nucleic acids to phospholipids, our data suggest that these enzymes are likely to utilize a common catalytic mechanism in which a
phosphohistidine enzyme intermediate is formed and subsequently hydrolyzed in a two-step reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dipalmitoyl[choline-methyl-3H]phosphatidylcholine
(50 Ci/mmol),
L-
-[myo-inositol-2-3H]phosphatidylinositol
(PI; 11 Ci/mmol);
[inositol-2-3H]phosphatidylinositol
4,5-bisphosphate (37 Ci/mmol),
L-
-dipalmitoyl-[2-palmitoyl-9,10-3H]phosphatidylcholine
(89 Ci/mmol), and
L-
-dipalmitoyl[glycerol-U-14C]phosphatidic
acid (PA; 100-200 Ci/mmol) were purchased from NEN Life Science
Products.
L-1,2-Dioleoyl-3-phosphatidyl[2-14C]ethanolamine
(55 mCi/mmol) and
L-1,2-dioleoyl-3-phosphatidyl-L-[3-14C]serine
(54 mCi/mmol) were purchased from Amersham Pharmacia Biotech. Silica
Gel 60 thin-layer chromatography plates were obtained from Alltech. All
other chemicals were of the highest quality available.
-D-thiogalactoside to a final concentration of 0.4 mM, and grown for an additional 4 h at 25 °C. The
cells were harvested by centrifugation at 5000 × g for
5 min, and the resulting pellets were frozen at
70 °C. Pellets
were thawed at 0 °C, resuspended in 20 ml of lysis buffer (30 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol,
0.1% Triton X-100), and lysed at 4 °C by three passages through a
French press at 1200 p.s.i. Cellular debris was removed by
centrifugation at 20,000 × g for 20 min at 4 °C.
The clarified supernatant was incubated with gentle rotation for 1 h at 4 °C with 12 ml of nickel nitrilotriacetic acid-agarose that
had been equilibrated in low salt buffer (30 mM Tris, pH
7.4, 150 mM NaCl, 10% glycerol). The slurry was poured into a 50-ml column and washed with high salt wash buffer (30 mM Tris, pH 6.8, 300 mM NaCl, 10% glycerol).
Ymt was eluted with a gradient of 0-200 mM imidazole.
Samples of fractions were subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), and fractions containing
a single band corresponding to Ymt were pooled and concentrated using a
Centriprep-30 filtration unit (Amicon), adjusted to 50% glycerol, and
stored at
20 °C. The final product was
99% pure by SDS-PAGE.
The concentration was calculated using the determined extinction
coefficient, 130,951.9 M
1 cm
1
(see below).
1 cm
1, and corrected
for pNP ionization (18). All reactions were linear for the indicated
time, and rates were corrected for spontaneous substrate hydrolysis.
-dipalmitoyl-[2-palmitoyl-9,10-3H]
phosphatidylcholine (89 Ci/mmol) with unlabeled phosphatidylcholine to
give a final concentration of 342 µM phosphatidylcholine
(0.12 µCi/nmol). 10 µM of wild-type or mutant enzyme
was incubated with 25 µl of vesicles for 1 h in a final volume
of 150 µl. Lipids were extracted from the mixture by the addition of
chloroform:methanol:acetic acid (50:50:0.5). The lower phase was dried
under nitrogen, dissolved in chloroform:methanol:acetic acid
(90:10:10), and subjected to thin-layer chromatography on a silica gel
60 plate in the solvent used for solubilization. Plates were sprayed
with En3HanceTM (NEN Life Science Products), and
radiolabeled products were detected by autoradiography. Radiolabeled
phosphatidic acid was used as a standard.
-dipalmitoyl[2-palmitoyl-9,10-3H]phosphatidylcholine
and unlabeled dipalmitoylphosphatidylcholine. Wild-type or mutant
enzyme (final concentration, 10 µM) was incubated with 25 µl of vesicles for 1 h in a final volume of 150 µl with or
without ethanol (final concentration, 0.5%). Lipids were extracted from the reaction by the addition of chloroform:methanol:acetic acid
(50:50:0.5). The lower phase was dried under nitrogen, dissolved in
chloroform:methanol:acetic acid (90:10:10), and subjected to thin-layer
chromatography as described above. Plates were sprayed with
En3HanceTM, and radiolabeled products were detected by
autoradiography. Radiolabeled phosphatidic acid and phosphatidylethanol
were used as standards.
-mercaptoethanol, 0.02% bromphenol blue) was added,
and the proteins were separated by SDS-PAGE in a 10% gel, which was
subsequently dried and autoradiographed. To evaluate the stability of
the phosphoenzyme, wild-type Ymt was labeled for 10 min at 25 °C as
described, quenched, subjected to SDS-PAGE, and transferred to
Immobilon P membrane (Millipore) in transfer buffer (250 mM
Tris, pH 8.3, 192 mM glycine, 20% methanol). Membranes
were rinsed with methanol and water, then incubated at 37 °C for 45 min with 200 mM Tris, pH 7.4, 1 M HCl, pH 1, 1 M NaOH, pH 13, or 200 mM Tris, pH 7.4, containing 1 M hydroxylamine as described previously (19).
Membranes were rinsed with water, dried, autoradiographed, and
subsequently stained with Coomassie Blue. For pH stability studies,
membranes containing phosphoprotein were counted in a Beckman
scintillation counter and subsequently incubated at the indicated pH at
37 °C for 45 min (20). Buffers used were as follows: pH 1.0, 1 M HCl; pH 2-6, 2 M sodium acetate; pH 7-10, 1 M Tris. After incubation, membranes were rinsed with water,
dried, counted, and stained with Coomassie Blue to verify that protein
remained bound. The extent of hydrolysis is reported as the percentage
of the initial radioactivity on the membrane.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
99% purity (Fig. 1B). Each liter of culture
yielded 10-25 mg of purified Ymt, and amino-terminal sequencing of the
recombinant protein revealed the predicted sequence,
Met-Leu-Gln-Ile-Asp-Asn.
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Fig. 1.
PLD superfamily members and purification of
Ymt. A, shown are three members of the PLD superfamily.
Nuc has one copy of the conserved motif
HXKX4DX6G(G/S),
whereas Ymt and PLD have two copies. Nuc also contains a signal
sequence represented by the hatched bar. B,
proteins were diluted in sample buffer containing -mercaptoethanol,
separated using a 10% acrylamide gel, and visualized with Coomassie
staining. Lane 1, pre-induction cell lysate; lane
2, post-induction cell lysate; lane 3, post-French
press supernatant; lane 4, 3 µg of nickel nitrilotriacetic
acid-agarose column eluate.
-dipalmitoyl[2-palmitoyl-9,10-3H]phosphatidylcholine
to form phosphatidic acid and, in the presence of 0.5% ethanol, the
transphosphatidylation product, phosphatidylethanol (data not shown).
Although Ymt displays PLD characteristics, hydrolysis of
phosphatidylcholine in either reaction appears not to be stimulated by
recombinant human ADP-ribosylation factor 1 (ARF1),
phosphatidylinositol 4,5-bisphosphate, or sodium oleate (data not
shown).
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Fig. 2.
Phospholipid headgroup release by Ymt.
Enzyme (final concentration, 5 µM) or an equal volume of
buffer was incubated with headgroup-labeled phosphatidylcholine
(PC), phosphatidylethanolamine (PE), or
phosphatidylserine (PS) (final concentration, 10 µM) as described under "Experimental Procedures."
Lipids were extracted in 1 ml of chloroform:methanol:concentrated HCl
(50:50:0.5) and 350 µl of 1 mM EGTA in 1 M
HCl; a portion of the aqueous layer was subjected to scintillation
counting. Reactions were performed in triplicate, and error
bars indicate the mean ± one-half the range.
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Fig. 3.
Bis(p-nitrophenyl) phosphate
hydrolysis by Ymt. Increasing concentrations (0-25
mM) of bis-pNPP were incubated with 1 µM Ymt
at 30 °C, and the absorbance of the released product, p-NP, was
monitored over 3 min (A). The resulting slopes were plotted
against the substrate concentration (B).
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Fig. 4.
Crystal of Ymt. Ymt crystals (size = 0.02 × 0.04 × 0.1 mm3) used in macroseeding
experiments are shown viewed under polarized light at 75×
magnification.
Bis(p-nitrophenyl) phosphate hydrolysis by wild-type and mutant Ymt
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Fig. 5.
SDS-PAGE analysis of Ymt mutants. Each
purified protein was diluted into sample buffer containing
-mercaptoethanol, and 3 µg was loaded onto a 10% acrylamide gel.
Lane 1, wild-type; lane 2, H188N; lane
3, H524N; lane 4, H188N/H524N; lane 5,
K190S; lane 6, K526S; lane 7, K190S/K526S;
lane 8, S539T; lane 9, S539A. The positions of
the molecular mass standards are indicated (kDa).
N). The data in Table I are most easily
explained if each dimer contains two active sites, and each active site
is composed of residues from two different domains. In model
A, the conserved motif in domain I of one monomer would
associate with the conserved motif in domain II of another monomer.
Substitution of a single histidine in either domain will affect the
catalytic activity at both active sites in the dimer, resulting in a
nearly complete functional loss (i.e. >99%). Model
C shows the monomers associating via the linker regions
between the domains. The active sites of the dimer in model
C are composed of residues found in the first and second
domains of a single monomer as opposed to domains from different
monomers as shown in model A. The data in Table I do not
afford discernment between these models. They do, however, rule out
model B, where conserved motifs in like domains form intermolecular interactions between monomers. Assuming that the catalytic activity is distributed equally between the two domains, model B would suggest that substitution of a single
histidine would result in only a 50% loss of activity, as one
functional intermolecular interaction between conserved residues would
be maintained.
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Fig. 6.
Three possible Ymt dimer configurations.
Each model represents a mutant Ymt dimer with a substitution
in the conserved histidine in one domain. Domains labeled H
contain a histidine in the conserved motif, and domains labeled
N have an asparagine substituted for the conserved
histidine. The amino (N) and carboxy (C) termini are indicated for each
monomer. A, two Ymt monomers associate to form heterodimers
such that histidines from different domains act in concert to form
active sites. B, homodimers form between Ymt monomers such
that histidines from like-domains interact to form active sites.
C, Ymt monomers associate through linker regions between
domains I and II such that intramolecular associations between
catalytic residues are formed.
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Fig. 7.
Possible mechanisms of Ymt catalysis. In
A, catalysis proceeds via a single-step displacement
mechanism where no intermediate is formed, i.e. Ymt acts as
a general base (Base-Ymt) to extract a proton from a
nucleophilic water molecule. In B, the reaction proceeds as
a two-step mechanism where Ymt serves as the nucleophile
(Nuc-Ymt). A phosphoenzyme intermediate is formed and
subsequently broken down by hydrolysis.
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Fig. 8.
Formation and properties of the phosphoenzyme
intermediate. A, 30 µM of bovine serum
albumin, wild-type Ymt (WT), or mutant Ymt was incubated
with 32P-inorganic phosphate in 200 mM sodium
acetate, pH 4.5. At the indicated times, aliquots of the reaction were
removed and quenched into 1 M
Na2CO3, pH 11, 0.5% SDS. Proteins were
separated by SDS-PAGE, and radioactivity was detected by
autoradiography. B, proteins were separated by SDS-PAGE and
transferred to Immobilon P membrane. Membranes were incubated for 45 min at 37 °C under the following conditions: lane 1,
buffer; lane 2, 1 M HCl; lane 3, 1 M NaOH; lane 4, 1 M hydroxylamine in
200 mM Tris, pH 7.4. Radiolabeled proteins were detected by
autoradiography (top panel), and the membranes were
subsequently stained with Coomassie Blue (bottom panel).
C, membranes with radiolabeled phosphoenzyme were incubated
for 45 min at 37 °C with 1 M HCl, pH 1, 2 M
sodium acetate, pH 2-6, or 1 M Tris, pH 7-10. Membranes
were counted before and after incubation, and the hydrolysis of the Ymt
phosphoprotein was calculated at each pH as a percentage of starting
counts on the membrane (19).
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Fig. 9.
Phosphoamino acid analysis. Complete
alkaline hydrolysis of radiolabeled Ymt was performed as
described under "Experimental Procedures." The resulting
radiolabeled amino acids and internal standards were resolved by
ion-exchange chromatography as described (20). The positions of
inorganic phosphate (Pi), phosphoarginine (R),
phospholysine (K), phosphothreonine (T),
phosphohistidine (H), and phosphotyrosine (Y)are shown.
Phosphoserine elutes 30 s after phosphothreonine (20).
Fluorescence is plotted on the right axis (solid
line), and radioactivity (data points) is plotted on
the left axis.
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Fig. 10.
Proposed mechanism of Ymt catalysis. In
the model for Ymt catalysis, the conserved histidine from one domain
(His-A) serves as the nucleophile. The conserved histidine
from a second domain (His-B) acts as a general acid to
protonate the leaving group, which facilitates the formation of a
covalent phosphoenzyme intermediate. The free histidine
(His-B) then functions as a general base to activate
water by proton abstraction. The intermediate is subsequently
hydrolyzed by the activated water molecule.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank K. A. Orth and C. A. Worby for critical reading of the manuscript, E. B. Gottlin for helpful discussions, and J. Zhou and K. Chan for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NIDDKD 18849 (to J. E. D.) and by the Walther Cancer Institute, Indianapolis, Indiana.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.
§ Present address: Monsanto/Searle, St. Louis, MO 63167. Supported in part by a Walther Cancer Institute postdoctoral fellowship.
¶ Supported by a National Institutes of Health (NIDDKD) National Research Service Award postdoctoral fellowship.
To whom correspondence should be addressed: University of
Michigan, Dept. of Biological Chemistry, M5416 Medical Science Bldg. I,
Ann Arbor, MI 48109-0606. Tel.: 734-764-8192; Fax: 734-763-6492; E-mail: jedixon{at}umich.edu.
2 B. J. Hinnebusch, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
PLD, phospholipase
D;
Ymt, Y. pestis murine toxin;
PAGE, polyacrylamide gel
electrophoresis;
pNPP, para-nitrophenyl phosphate;
pNP, para-nitrophenolate;
PI, L--[myo-inositol-2-3H]phosphatidylinositol;
PA, L-
-dipalmitoyl[glycerol-U-14C]phosphatidic
acid.
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REFERENCES |
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