From the Atlantic Research Centre, Departments of Pediatrics and
Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H
4H7, Canada
Cholinephosphotransferase (EC 2.7.8.2) catalyzes
the formation of a phosphoester bond via the transfer of a
phosphocholine moiety from CDP-choline to diacylglycerol forming
phosphatidylcholine and releasing CMP. A motif,
Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127-(X)3-Asp131-(X)3-Asp135,
located within the CDP-choline binding region of Saccharomyces cerevisiae cholinephosphotransferase (CPT1 ?/Author:
Please confirm that a gene is meant here.) is also found in several
other phospholipid synthesizing enzymes that catalyze the formation of
a phosphoester bond utilizing a CDP-alcohol and a second alcohol as
substrates. To determine if this motif is diagnostic of the above
reaction type scanning alanine mutagenesis of the conserved residues
within S. cerevisiae cholinephosphotransferase was
performed. Enzyme activity was assessed in vitro using a
mixed micelle enzyme assay and in vivo by determining the
ability of the mutant enzymes to restore phosphatidylcholine synthesis
from radiolabeled choline in an S. cerevisiae strain devoid
of endogenous cholinephosphotransferase activity. Alanine mutants of
Gly114, Gly127, Asp131, and
Asp135 were inactive; mutants, Ala117 and
Arg118 displayed reduced enzyme activity, and
Asp113 displayed wild type activity. The analysis described
is the first molecular characterization of a CDP-alcohol
phosphotransferase motif and results predict a catalytic role utilizing
a general base reaction proceeding through Asp131 or
Asp135 via a direct nucleophilic attack of the hydroxyl of
diacylglyerol on the phosphoester bond of CDP-choline that does not
proceed via an enzyme bound intermediate. Residues Ala117
and Arg118 do not participate directly in catalysis but are
likely involved in substrate binding or positioning with
Arg118 predicted to associate with a phosphate moiety of
CDP-choline.
 |
INTRODUCTION |
Cholinephosphotransferase catalyzes the transfer of phosphocholine
from CDP-choline to diacylglycerol
(DAG)1 thus forming
phosphatidylcholine (PtdCho) and CMP. As the final step of the Kennedy
pathway (1, 2) cholinephosphotransferase identifies both the site of
de novo PtdCho synthesis as well as the site from which
PtdCho is transported to other organelles within the cell or assembled
with proteins and other lipids for export from the cell during the
genesis of lung surfactant, bile, and lipoproteins (3-5). Two genes
coding for cholinephosphotransferase activities exist in
Saccharomyces cerevisiae, CPT1, which encodes a
cholinephosphotransferase (6), and EPT1, which codes for a
dual specificity choline/ethanolaminephosphotransferase (7). In
vitro, Cpt1p and Ept1p contribute equally to measurable
cholinephosphotransferase activity (8); however, in vivo
metabolic labeling analysis revealed that Cpt1p is responsible for 95%
of the PtdCho-synthesizing cholinephosphotransferase activity with
Ept1p contributing the final 5% (9). Both CPT1 and
EPT1 predict proteins containing seven membrane spanning
domains. The integral membrane bound nature of
cholinephosphotransferase has prevented its purification from any
source and has precluded the use of many standard structure/function approaches for analyzing S. cerevisiae Cpt1p and Ept1p
enzymes. However, the uninterrupted similarity in predicted secondary
structures and corresponding nucleic acid sequences allowed for the
construction of a series of CPT1/EPT1-encoded chimeric
enzymes (10, 11). The difference in CDP-alcohol specificity between the
parental Cpt1p and Ept1p enzymes was exploited to delineate the
CDP-choline binding site. A region encompassing the first soluble loop,
residues 79-186 of Cpt1p, was pinpointed. Data base searches for known proteins with homology to the CDP-choline binding region of Cpt1p identified sequences within: S. cerevisiae
ethanolaminephosphotransferase (EPT1) (7),
phosphatidylinositol (PtdIns) synthase (PIS1) (12), and
phosphatidylserine (PtdSer) synthase (PSS1/CHO1)
(13); prokaryotic phosphatidylglycerol (PtdGro) phosphate and PtdSer
(Gram-positive only) synthases (14, 15); soybean
aminoalcoholphosphotransferase (16); and rat PtdIns synthase (17). Each
of these enzymes catalyzes the synthesis of a phospholipid by the
displacement of CMP from a CDP-alcohol by a second alcohol to form a
phosphoester bond. Alignment of the sequences within each of the above
enzymes revealed a completely conserved motif,
Asp-Gly-(X)2-Ala-Arg-(X)8-Gly-(X)3-Asp-(X)3-Asp, termed the CDP-alcohol phosphotransferase motif. Data base searches revealed this motif was specific to the above enzymes and hence is
predicted to be diagnostic for the reaction type catalyzed by
each. To lend credence to this prediction, and to provide insight into
catalytic mechanism, scanning alanine mutagenesis of the conserved
residues within the CDP-alcohol phosphotransferase motif of S. cerevisiae cholinephosphotransferase (CPT1) was
performed.
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EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]dATP (3000 Ci/mmol) and
[methyl-14C]choline were products of NEN Life
Science Products. [methyl-14C]CDP-choline was
purchased from American Radiolabeled Chemicals. Custom oligonucleotides
were purchased from Life Technologies, Inc. Dideoxy sequencing was
performed utilizing the T7 sequencing kit (Amersham Pharmacia Biotech).
Lipids were purchased from Avanti Polar Lipids. Triton X-100 was
purchased from Pierce. Hemagglutinin (HA) monoclonal antibody (mAb) was
purchased from Boehringer Mannheim. All other reagents were of the
highest quality commercially available.
Site-directed Mutagenesis--
Plasmid pRH150 (6) contains the
CPT1 gene in the high copy plasmid YEp352 (18) and was used
as the template for all mutagenesis reactions. The T4 DNA
polymerase-directed MORPH plasmid DNA mutagenesis protocol (5'
3')
was used with the appropriate mutagenic oligonucleotides (Table
I) as directed by the manufacturer. All
mutations were confirmed by DNA sequencing. Plasmids were routinely
propagated in DH5
Escherichia coli (endA1
recA1 hsdR17
(rk
mk+) supE44
thi-1 gyrA(NaIr) relA1
deoR (
80lacZ
M15)
(lacZYA-argF)U169.
Enzyme Assays--
S. cerevisiae strain HJ091
(a ura3-52 his3-1 leu2-3, 112 trp1-289
cpt1::LEU2 ept1
) was utilized in all
studies. HJ091 is devoid of detectable cholinephosphotransferase activity (10, 11). Microsomal membranes were prepared from HJ091 grown
at 30 °C to mid-log phase in synthetic dextrose media containing the
appropriate nutritional supplements to ensure plasmid maintenance (19).
Cholinephosphotransferase activity was assessed using 20 mM
MgCl2 as cation cofactor, 10 mol % PtdCho (egg) as phospholipid cofactor, and 10 mol % dipalmitoleoylglycerol (di16:1DAG) and 500 µM [14C]CDP-choline (500-4000
dpm/nmol as dictated by the sensitivity required) as substrates in a
Triton X-100 mixed micelle assay as described (9).
PtdCho Biosynthesis--
Yeast strain HJ091 ± parental and
mutagenized plasmids were grown to mid-log phase in synthetic dextrose
media containing the appropriate nutritional supplements.
[14C]Choline (20 µM, 1 × 105 dpm/nmol) was added to the cultures for 30 min. Yeast
cells were concentrated by centrifugation, washed with ice-cold water,
and resuspended in 1 ml of CHCl3/CH3OH (1/1,
v/v). Cells were disrupted using a BioSpec multibead beater containing
0.5 g of 0.5-mm acid washed glass beads on the homogenize setting
for 1 min at 4 °C. The beads were washed with
CHCl3/CH3OH (2/1, v/v). A sample was removed
from the total homogenate for determination of total choline uptake. To
facilitate phase separation, water and CHCl3 were added to
the remaining homogenate. Phospholipids in the organic phase were
analyzed by thin layer chromatography on Whatman silica gel 60A plates
using the solvent system
CHCl3/CH3OH/NH4OH/H20
(70/30/4/2, v/v). PtdCho was the only radiolabeled lipid detected.
Aqueous metabolites were concentrated under vacuum, resuspended in
H2O, and separated by thin layer chromatography on Whatman
silica gel 60A plates in a solvent system consisting of
CH3OH/0.6% NaCl/NH4OH (50/50/5, v/v). Choline,
phosphocholine, and CDP-choline were the only radioactive metabolites
detected. Radiolabel was detected using a Bioscan System 200 imaging
scanner and the radioactive bands were scraped into vials and subjected
to scintillation counting.
Immunodetection--
The CPT1 gene and site-directed
mutants were subcloned from YEp352 (URA3, 2 µ ori] to pRS426 [URA3,
2 µ ori] to facilitate the elimination of a HindIII site
within the multicloning site. A 3× repeat of the HA epitope was
amplified by polymerase chain reaction from the plasmid pGTEP (gift
from Stephen Garrett, Duke University Medical Center) with
HindIII sites added to the ends of each primer. The
amplified 3× HA epitope was subcloned into a unique HindIII
site within the CPT1 coding region corresponding to amino
acids Tyr28 and Leu29. The insert was sequenced
to ensure polymerase frame and fidelity. HJ091 cells were transformed
with the constructs and microsomal membranes were isolated. Identical
(10 µg) amounts of microsomal protein were incubated with equal
volumes of 2 × SDS sample buffer (50 mM Tris-HCl (pH
6.8), 10% glycerol (v/v), 2% SDS (w/v), 5% 2-mercaptoethanol (v/v),
0.05% bromphenol blue (w/v)) at 37 °C for 20 min. Proteins were
separated by SDS-PAGE (4% stacking gel, 12.5% resolving gel), and
transferred to polyvinylidene fluoride membrane utilizing the Bio-Rad
minigel transfer apparatus at 30 volts for 18 h in 48 mM Tris, 30 mM glycine, 0.1% SDS, 20%
methanol (v/v) transfer buffer. The membrane was: blocked with
phosphate-buffered saline (PBS) containing 5% skim milk powder and
0.05% Tween 20 for 1 h; washed three times with PBS, 0.05% Tween
20; incubated for 1 h in PBS, 0.05% Tween 20 with HA mAb
(1:2,000); washed three times with PBS, 0.05% Tween 20; incubated for
1 h in PBS, 0.05% Tween 20 with goat anti-mouse Ab coupled to
horseradish peroxidase (1:10,000); washed three times with
PBS, 0.05% Tween 20; and signal was detected using the ECL
(Amersham) method as per the manufacturer's instructions.
Protein and Lipid Determinations--
Protein was determined by
the method of Lowry et al. (20) using bovine serum albumin
as standard. DAG was prepared from PtdCho by phospholipase C digestion,
and yield was estimated using the method of Stern and Shapiro (21).
Phospholipid phosphorus was determined by the method of Ames and Dubin
(22).
 |
RESULTS |
Enzyme Activity of the Cpt1p CDP-alcohol Phosphotransferase
Mutants--
Scanning alanine site-directed mutagenesis of the
CDP-alcohol phosphotransferase motif
(Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127-(X)3-Asp131-(X)3-Asp135,
the one Ala residue was converted to Gly) of S. cerevisiae
Cpt1p was performed to lend credence to the prediction that this motif is diagnostic for the reaction type catalyzed by this class of enzymes,
which also includes: S. cerevisiae
ethanolaminephosphotransferase (EPT1) (7), PtdIns synthase
(PIS1) (12), and PtdSer synthase (PSS1/CHO1) (13), prokaryotic PtdSer synthase and
PtdGro phosphate synthase (14, 15), soybean
aminoalcoholphosphotransferase (16), and rat PtdIns synthase (17) (Fig.
1). The CDP-alcohol phosphotransferase
motif of Cpt1p was chosen as the target motif for several reasons: (i)
a well established mixed micelle assay exists enabling kinetic analysis
of enzyme activity (8, 10, 23); (ii) Cpt1p is a well characterized
member of the known CDP-alcohol phosphotransferase enzymes and coupled
with S. cerevisiae mutants devoid of
cholinephosphotransferase activity result in an effective and
established expression system (8, 9, 24); (iii) the metabolic fate of
radiolabeled choline for PtdCho synthesis has been rigorously
characterized in S. cerevisiae with defects in
cholinephosphotransferase activity allowing for in vivo
corroboration of in vitro results (9, 24). The
CPT1 gene was carried on the high copy plasmid YEp352 and
was used for all mutagenesis and expression experiments.
CPT1 is utilizing its own promoter in all analyses.
Mutagenesis of each Ala residue was represented by the preferred GCT
codon to ensure against codon bias during mRNA translation
(25).

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Fig. 1.
Known enzymes possessing a CDP-alcohol
phosphotransferase motif. Footnote a, other prokaryotic homologues
catalyzing the same reaction also contain the motif.
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Specific enzyme activities were determined using a Triton X-100 based
mixed micelle assay (8, 10, 23) under optimal reaction conditions for
the wild type enzyme (Table II).
Conversion of Asp113 to Ala did not appreciably effect
enzyme activity; however, Ala117 to Gly, and
Arg118 to Ala replacements decreased activity to 17.8% and
10.1% wild type levels. Separate conversion of Gly114 to
Ala as well as each of the final three amino acids within the motif,
Gly127, Asp131, and Asp135, to Ala
eliminated detectable cholinephosphotransferase activity. Increasing
the sensitivity of the assay by increasing the specific radioactivity
of the substrate 8-fold coupled with 4-fold additional protein, a
32-fold total increase in activity detectability over standard assay
conditions, did not result in radiolabeled PtdCho production,
indicating these mutations effectively eliminated enzyme activity.
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Table II
Cholinephosphotransferase activity of Cpt1p CDP-alcohol
phosphotransferase motif mutants
Enzyme activities were determined in the microsomal membrane fraction
of strain HJ091 (cpt1::LEU2 ept1) harboring the
high copy plasmid pRH150[CPT1] containing the indicated
point mutations using the mixed micelle assay as described under
"Experimental Procedures."
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In Vivo Analysis of the Cpt1p CDP-alcohol Phosphotransferase
Mutants--
To corroborate the above in vitro assessment
of the ability of the various CDP-alcohol phosphotransferase motif
site-directed mutants to confer cholinephosphotransferase activity,
each mutant was expressed in S. cerevisiae cells devoid of
cholinephosphotransferase and their capacity to incorporate
radiolabeled choline into PtdCho was determined (Fig.
2). Since the rate-limiting step for
PtdCho synthesis is normally at the level of phosphocholine
cytidylyltransferase, alterations in cholinephosphotransferase activity
do not generally affect the ability of cells to incorporate
radiolabeled choline into PtdCho (9, 24, 26, 27). In addition, the
metabolic labeling protocol is much more sensitive than the in
vitro enzyme assay and does not rely on the ability of the various
mutant enzymes to survive cell disruption and subcellular fractionation
and hence provides a second level of confidence for determining if
mutants with undetectable cholinephosphotransferase activity in
vitro are indeed devoid of enzyme activity. The enzymatically
active Asp113 to Ala, Ala117 to Gly, and
Arg118 to Ala mutants incorporated labeled choline into
PtdCho at a level similar to that of cells carrying the parental Cpt1p
protein. The Gly114 to Ala, Gly127 to Ala,
Asp131 to Ala, and Asp135 to Ala mutants, each
of which was inactive in vitro, were unable to incorporate
radiolabeled choline into PtdCho (Fig. 2B). In agreement
with a metabolic block at the level of cholinephosphotransferase in the
S. cerevisiae cells used in this study
(cpt1::LEU2 ept1
), this yeast strain
(and each of the inactive Cpt1p mutants) accumulated CDP-choline (Fig.
2C), while the wild type cells (and each of the mutants with
detectable cholinephosphotransferase activity in vitro)
did not accumulate CDP-choline due to its successful conversion to
PtdCho (Fig. 2, B and C).

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Fig. 2.
Radiolabeled choline uptake and incorporation
into the metabolites of the CDP-choline pathway. Exponentially
growing S. cerevisiae cells (cpt1::LEU2
ept1 ) containing CPT1 CDP-alcohol phosphotransferase
motif site-directed mutants were radiolabeled with 20 µM
choline for 30 min as described under "Experimental Procedures."
A, the Kennedy pathway for PtdCho synthesis; B,
total uptake of radiolabel and its incorporation into PtdCho;
C, the accumulation of radiolabeled choline within the
metabolites of the Kennedy pathway. Results are the mean of two
experiments performed in duplicate; variation was less than 10%
between experiments.
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Kinetic Analysis of the Cpt1p CDP-alcohol Phosphotransferase
Mutants--
As a first step in determining catalytic mechanism for
cholinephosphotransferase, a kinetic analysis of each of the
enzymatically active mutants was performed and compared with the
parental enzyme. The mixed micelle assay employed revealed saturation
kinetics for both substrates when the other was in excess (data not
shown) implying surface dilution of the lipophilic substrate DAG occurs within the mixed micelle and hence the kinetic parameters reported here
are reflective of true values. Assay buffer contained 500 µM CDP-choline and 10 mol % di16:1 DAG, and under these
conditions wild type Cpt1p displayed an apparent Km
of 66 µM for CDP-choline and 1.75 mol % for DAG (Tables
III and IV). Previous measurements of these
same parameters for Cpt1p revealed apparent Km
values of 110 µM and 8.0 mol % for CDP-choline and DAG,
respectively (8). However, the values obtained in the current study are
predicted to be more accurate, since the DAG substrate used in the
former study (8) was di18:1 DAG, and it has since been discovered that
di16:1 DAG is the preferred substrate for Cpt1p (10). In addition,
di18:1 DAG is insoluble at concentrations above 10 mol % in the mixed
micelle assay employed in both studies and hence was previously used at
suboptimal levels for the accurate measurement of kinetic parameters.
Di16:1 DAG does not present this problem as it is soluble up to 20 mol
%. The measured apparent Km value of 1.75 mol % for di16:1 DAG is 5.7-fold lower than the concentration of di16:1 DAG
(10 mol %) utilized in this study.
Kinetic parameters were also determined for the three mutants,
Asp113 to Ala, Ala117 to Gly, and
Arg118 to Ala, that possessed cholinephosphotransferase
activity. The Asp113 to Ala mutant displayed specific
activity and kinetic values similar to those of the parental enzyme
with apparent Km values of 55 µM for
CDP-choline and 2.28 mol % for DAG (Tables III and IV). Specific
enzyme activity measured for Ala117 to Gly and
Arg118 to Ala decreased from wild type levels of 6.021 nmol
min
1 mg
1 down to 1.022 nmol
min
1 mg
1 and 0.576 nmol min
1
mg
1, respectively (Table II). The Ala117 to
Gly mutation increased the apparent Km for
CDP-choline 2.5-fold and for DAG 2.8-fold over those of the parental
enzyme. The Arg118 to Ala mutation resulted in increased
apparent Km values of 3.8-fold for CDP-choline and
3.1-fold for DAG. Predicted Vmax values were
1.01 and 1.21 nmol min
1 mg
1 for the
Ala117 to Gly mutant, and 0.60 and 0.55 nmol
min
1 mg
1 for the Arg118 to Ala
mutant (Tables III and IV).
Immunodetection of Parental and Mutant
Cholinephosphotransferases--
In this study, parental and mutant
CPT1 genes were expressed from high copy (URA3, 2 µ ori)
plasmids using the endogenous CPT1 promoter. Western blot
analysis was performed to ensure that the various mutations created
within Cpt1p (with activities significantly different from wild type)
were not affecting Cpt1p levels. A 3× repeat of the HA epitope was
inserted into the coding region of the parental and site-directed
mutant genes at a site between amino acids Tyr28 and
Leu29. Plasmids were transformed into HJ091 cells and
microsomal membrane proteins were subjected to SDS-PAGE and Western
blot analysis using mAb specific for the HA epitope (Fig.
3). The HA mAb recognized a protein of
the expected size for Cpt1p+3× HA epitope (49 kDa) that was absent in
cells expressing Cpt1p that did not contain the 3× HA epitope,
demonstrating that the mAb was specific for epitope tagged Cpt1p
proteins (the increased size of Ala117 is a function of the
cloning strategy utilized that resulted in a 6× insertion of the HA
epitope). Parental and mutant Cpt1p proteins Ala117 to Gly,
Arg118 to Ala, Gly127 to Ala,
Asp131 to Ala, and Asp135 to Ala were present
in similar amounts (Fig. 3) confirming that the decreased
cholinephosphotransferase enzyme activity associated with the amino
acid substitutions of these residues was due to altered kinetic
properties of the mutant proteins and not due to decreased protein
levels. No Cpt1p protein was detected for the Gly114 to Ala
mutant, indicating that the absence of cholinephosphotransferase activity associated with this amino acid substitution was due to
increased protein lability.

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Fig. 3.
Immunodetection of parental and site-directed
mutants of Cpt1p. Microsomal membranes were isolated from HJ091
cells expressing parental and mutant forms of 3× HA epitope-tagged
Cpt1p. Equivalent amounts of microsomal protein were subjected to
SDS-PAGE and analyzed by Western blot utilizing mAb specific for the HA
epitope as described under "Experimental Procedures."
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 |
DISCUSSION |
Scanning alanine site-directed mutagenesis of the conserved amino
acid residues within the CDP-alcohol phosphotransferase motif
(Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127-(X)3-Asp131-(X)3-Asp135)
of S. cerevisiae cholinephosphotransferase was performed to lend credence to the prediction that this motif is diagnostic for the
reaction catalyzed and to provide the first molecular investigation of
the catalytic mechanism used by this class of enzymes which to date
include: S. cerevisiae cholinephosphotransferase, ethanolaminephosphotransferase, PtdSer synthase, PtdIns synthase, prokaryotic PtdSer synthases, PtdGro phosphate synthases, and rat
PtdIns synthase. Each of these enzymes catalyzes the formation of a
phospholipid via the displacement of CMP from a CDP-alcohol by a second
alcohol with concomitant formation of a phosphoester bond. The ability
of each mutant to catalyze the cholinephosphotransferase reaction was
assessed in vitro using a mixed micelle enzyme assay and
in vivo by determining the capacity of each mutant to
incorporate radiolabeled choline into PtdCho in a S. cerevisiae strain containing null mutations in its
cholinephosphotransferase genes.
One mutant, Asp113 to Ala, displayed wild type
characteristics both in vitro and in vivo. This
is intriguing in that this, the first Asp residue within the
CDP-alcohol phosphotransferase motif, is completely conserved over a
wide range of species and hence has not drifted evolutionarily implying
it is essential; however, its function is not apparent from the above
analyses. Two mutants, A117 to G and R118 to A,
displayed reduced in vitro catalytic activity due to
5.0-10.4-fold decreased Vmax(app) and
2.5-3.8-fold increased Km(app) values for both
CDP-choline and DAG. These results imply Ala117 and
Arg118 play a role in substrate binding or positioning with
Arg118 predicted to be coordinated with one of the
phosphate groups within the CDP-alcohol. Interestingly, in
vivo the Ala117 to Gly and Arg118 to Ala
mutants incorporated radiolabeled choline into PtdCho in
cholinephosphotransferase null cells at levels similar to that of the
parental enzyme. These data support two further conclusions: (i) the
concentration of both CDP-choline and DAG at the intracellular site of
cholinephosphotransferase must be sufficient to overcome the increase
in Km(app) demonstrated by the Ala117 to
Gly and Arg118 to Ala mutants, and (ii)
cholinephosphotransferase is not rate-limiting, since large decreases
in Vmax(app) did not affect the level with which
labeled choline was incorporated into PtdCho. The latter conclusion is
consistent with many other metabolic studies (24, 26). Mutations at
Gly114, Gly127, Asp131, and
Asp135, completely ablated detectable activity both
in vitro and in vivo. The absence of
cholinephosphotransferase activity in cells expressing Cpt1p carrying a
Gly114 to Ala substitution correlated with an absence of
detectable protein, indicating this residue is required for protein
stability or folding. Since Gly residues do not possess a functional
group, the lack of activity in the Gly127 to Ala mutant
suggests a steric role within the active site. The elimination of
cholinephosphotransferase activity by mutating either
Asp131 or Asp135 (the last two residues within
the motif) to Ala implies one of these is the catalytic residue.
Several members of the CDP-alcohol phosphotransferase motif family of
enzymes have been subjected to kinetic analyses; pure enzyme
preparations of S. cerevisiae PtdSer synthase and E. coli PtdGro phosphate synthase, as well as microsomal mammalian
cholinephosphotransferase, predicted sequential bi-bi reaction
mechanisms (28-31). An in depth kinetic analysis of purified E. coli PtdGro phosphate synthase was consistent with a reaction
mechanism that did not utilize an enzyme bound intermediate. A general
acid-catalyzed reaction utilizing an Asp residue at its catalytic
center would favor an enzyme bound intermediate, while a general base
reaction would not, hence, CDP-alcohol phosphotransferases most likely
utilize general base catalysis via nucleophilic attack of the hydroxyl group of the free alcohol directly on the phosphoester bond of the
CDP-alcohol through one of the final two Asp residues within the motif
without passing through an enzyme-bound intermediate.
From the above results it is clear that the CDP-alcohol
phosphotransferase motif,
Asp-Gly-(X)2-Ala-Arg-(X)8-Gly-(X)3-Asp(X)3-Asp, is diagnostic for the reaction catalyzed. However, it should be noted that this motif is not essential for this reaction type. E. coli PtdSer synthase catalyzes the formation of PtdSer via formation of a phosphoester bond utilizing CDP-DAG and serine as
substrates with subsequent release of CMP in a manner similar to that
of the Bacillus subtilus and S. cerevisiae PtdSer
synthases (31-33). The latter two possess the CDP-alcohol
phosphotransferase motif while the E. coli enzyme possesses
a separate motif, HxK(U)4D(X)4UUGO, that also
appears capable of the formation of the identical phosphoester bond
(34, 35). This second motif is also found in prokaryotic cardiolipin
synthase, as well as enzymes that catalyze the hydrolysis of a
phosphoester bond including phospholipase D and some nucleases. Studies
of E. coli and S. cerevisiae PtdSer synthases
provide two further lines of evidence that are consistent with two
distinct motifs catalyzing the same reaction via different mechanisms: (i) kinetic analysis of E. coli PtdSer synthase predicted a
ping-pong reaction type that utilized an enzyme-bound intermediate
(31-33), a mechanism distinct from the sequential bi-bi reaction of
CDP-alcohol phosphotransferase motif enzymes (28-31), and (ii) an
isotopic exchange NMR analysis comparing the PtdSer synthase activities from E. coli and S. cerevisiae observed a
retention of chirality of the
-phosphorus within the
CDP-diacylglycerol substrate for the E. coli enzyme
(consistent with a reaction mechanism proceeding via an enzyme-bound
intermediate), while the S. cerevisiae enzyme displayed an
inversion of chirality of the
-phosphorus (implying a single
displacement mechanism) (31).
This study is the first molecular characterization of a CDP-alcohol
phosphotransferase motif and results indicate the motif plays an
intimate role in catalysis. Several lines of evidence point to this
conclusion: (i) the CDP-alcohol phosphotransferase motif is completely
conserved in enzymes that catalyze the same reaction type; (ii) the
conservation is observed across a wide range evolutionary range
implying the preserved residues are essential for enzyme function;
(iii) FASTAPAT and Motif searches of nonredundant data bases revealed
only the above enzymes indicating its specificity; (iv) mutations
within specific residues of the S. cerevisiae
cholinephosphotransferase CDP-alcohol phosphotransferase motif abolish
or reduce activity. The integral membrane-bound nature of all of the
members of the CDP-alcohol phosphotransferase motif enzymes has
precluded their purification from most sources, including any mammalian
cell type, thus many of their respective cDNAs have yet to be
isolated. The verification of a conserved motif diagnostic for the
reaction type catalyzed by each of these enzymes will allow for the
rapid identification of cDNAs coding for these proteins from
expressed sequence tag data bases. These new molecular tools will allow for a precise dissection of the many biological and pathophysiological roles currently postulated for each of these enzymes.