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PHOSPHATIDYLSERINE SYNTHASEFROME. coli
287
[34] P h o s p h a t i d y l s e r i n e S y n t h a s e f r o m E s c h e r i c h i a coli By WILLIAM DOWHAN
Introduction (d)CDPdiacylglycerol + L-serine---> phosphatidylserine + (d)CMP
Phosphatidylserine synthases (EC 2.7.8.8, CDPdiacylglycerol-L-serine O-phosphatidyltransferase) from several gram-negative bacteria have been partially characterized and seem to be very similar in their physical and catalytic propertiesl'2; the enzyme from Escherichia coli has been extensively studied and characterized. Phospholipid biosynthetic enzymes in bacteria are generally found associated with membranes, but the phosphatidylserine synthases from gram-negative bacteria are found associated with the ribosomal fraction of cell lysates.l'2 The same reaction is catalyzed by the phosphatidylserine synthases from Saccharomyces cerevisiae 3 and the gram-positive bacilli, 4'5 but the enzymes from these organisms, unlike the enzymes from gram-negative bacteria, are dependent on added divalent metal ions for activity and are membrane associated. A similar CDPdiacylglycerol-dependent phosphatidylserine synthase activity has never been found in higher eukaryotic cells. Assay Method The enzyme is routinely assayed by following the incorporation of radiolabeled L-serine into chloroform-soluble material in the presence of a nonionic detergent such as Triton X-100; the ribose and deoxyribose forms of the liponucleotide are equivalent substrates. 6 In crude extracts either [3H]serine or [3-14C]serine should be used since the product is rapidly convened to phosphatidylethanolamine by the presence of the phosphatidylserine decarboxylase7; alternatively [U-14C]serine can be used if the decarboxylase is inhibited by inclusion of 10 mM hydroxylamine in the assay. Highly purified preparations of the enzyme can be assayed I A. Dutt and W. Dowhan, J. Bacteriol. 132, 159 (1977). 2 C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 247, 2008 (1972). 3 G. M. Carman and M. Bae-Lee, this volume [35]. 4 A. Dutt and W. Dowhan, J. Bacteriol. 147, 535 (1981). A. Dutt and W. Dowhan, Biochemistry 24, 1073 (1985). 6 T. J. Larson and W. Dowhan, Biochemistry 15, 5212 (1976). 7 W. Dowhan, W. T. Wickner, and E. P. Kennedy, J. Biol. Chem. 249, 3079 (1974).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SYNTHASES
[34]
spectrophotometrically by coupling the continuous release of CMP to the oxidation of NADH in the presence of ATP and phosphoenolpyruvate via the sequential actions of CMP kinase, pyruvate kinase, and lactate dehydrogenase.8'9 P r o c e d u r e . 6 If necessary the enzyme is diluted with 0.1 M potassium phosphate buffer (pH 7.4) containing 0.1% (1.6 mM) Triton X-100 and 1 mg/ml of bovine serum albumin (BSA). Dilution and assay are done in polypropylene tubes since the dilute, purified enzyme tends to adhere to glass; serum albumin and high protein concentrations minimize this problem. The final concentrations of the components of the assay mixture are 0.67 mM CDPdiacylglycerol (synthesized from egg yolk phosphatidylcholine as described elsewhere in this volumel°), 0.5 mM L-serine (radiolabeled as desired to a specific activity of 2-4/zCi//zmol), 0.1% Triton X-100, 0.1 M potassium phosphate buffer (pH 7.4), and 1 mg/ml BSA; an ionic strength of 0.3 or higher is optimal. 9 The reaction is initiated by adding 10/zl of an enzyme solution to 50/zl of a 1.2-fold concentrated stock solution of the above assay mixture. The reaction mixture is incubated in a 12-ml polypropylene tube at 30° for 10 min. The reaction is stopped by adding, in the following order, 0.5 ml of methanol (0.1 N in HC1), 1.5 ml chloroform, and 3.0 ml of 1 M MgCI2. After thorough mixing of the two phases and separation of the phases by centrifugation at 1000 gav in a clinical centrifuge at room temperature, 1 ml of the chloroform phase (lower phase) is removed, evaporated to dryness at 65°, and counted for radioactivity using any commercially available scintillation fluid. One unit of activity is defined as the amount of enzyme which converts 1 /zmol of L-serine into chloroform-soluble material in 1 min under the above conditions. The assay is linear for the pure enzyme up to about 50% conversion of the limiting substrate. When CDPdiacylglycerol is the limiting substrate, total conversion approaches only 95%, probably because of an inherent hydrolase activity which is 1% of the synthase rate. In crude preparations the linearity may fall off before reaching 50% conversion depending on the degree of contamination by the endogenous, membrane-associated CDP-diacylglycerol hydrolase activityll; this activity can be inhibited by 1 mM ATP.
8 G. M. Carman and W. Dowhan, J. Lipid Res. 19, 519 (1978). 9 G. M. Carman and W. Dowhan, J. Biol. Chem. 2,54, 8391 (1979). 10 G. M. Carman and A. S. Fischl, this volume [36]. u C. R. H. Raetz, C. B. Hirschberg, W. Dowhan, W. T. Wickner, and E. P. Kennedy, J. Biol. Chem.247, 2245 (1972).
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PHOSPHATIDYLSERINE SYNTHASEFROME. coli
289
TABLE I PURIFICATION OF PHOSPHATIDYLSERINE SYNTHASEa
Step I. 2. 3. 4.
Cell-free extract Cell supernatant Phosphocellulose DEAE-Sephadex
Total protein
Specific activity (units/mg)
Yield (%)
3.1 g 3.0 g ND b 34 mg
0.80 0.87 ND 39
100 104 70 54
a Starting with 20 g of cell paste from E. coli strain JA200/pPS3155-k as described by A. Ohta, K. Waggoner, K. Louie, and W. Dowhan, J. Biol. Chem. 256, 219 (1981). b Not determined.
Purification Procedure It is possible to obtain near gram quantities of highly purified enzyme by utilizing a purification scheme which relies on specific elution of the enzyme from phosphocellulose using the liponucleotide substrate 6 and starting with a strain of E. coli containing a plasmid-borne copy of the structural gene (pss) which directs the overproduction of the enzyme 100to 200-fold. 12The structural gene for the phosphatidylserine synthase has been introduced into a derivative of plasmid pBR322 (pPS3155-h) which carries the N O P region of h phage13; the N O P region contains the h origin of replication, a temperature-sensitive ~ repressor, and the essential promoters. At 30° this plasmid replicates from the normal pBR322 origin and is carried at about 11 copies per cell. Induction at 42° results in inactivation of the h repressor and an increase in the copy number of the plasmid to greater than 100 per cell. Because the production of the pss gene product is gene dose-dependent, cells carrying this plasmid grown at 42 ° have greatly elevated levels of phosphatidylserine synthase. Except where indicated the following steps (taken from Ohta et a l ) 2 and summarized in Table I) are carried out at 4 °. Growth of Cells. Cells can be grown in batches of 1 liter or less in shaking flasks or in 100-liter amounts in a fermentor using SB broth [12 g/ liter of Bacto-tryptone (Difco, Detroit, MI), 25 g/liter of yeast extract, 0.1 M potassium phosphate (pH 7.0), and 0.5% glucose] supplemented with 50/zg/ml ampicillin (the plasmid drug marker). Strain JA200/pPS3155-)t is grown to mid-log phase (OD550 1.5) at 30°, rapidly shifted to 42° for 30 min, and then incubated at 37° for 4 hr before harvesting the cells; high aeration and rapid shaking are maintained throughout the growth period. The yield 12 A. Ohta, K. Waggoner, K. Louie, and W. Dowhan, J. Biol. Chem. 256, 219 (1981). t3 R. N. Rao and S. G. Rogers, Gene 3, 247 0978).
290
SYNTHASES
[34]
of cells is about 12 g/liter of wet weight cells, with a specific activity for the phosphatidylserine synthase ranging between 100- and 200-fold over that from strain JA200 lacking the plasmid. The cell paste can be stored at - 8 0 ° for years. Step 1: Cell-Free Extract. Cell paste (20 g) of strain JA200/pPS3155-~ is suspended in 70 ml of buffer containing 0.1 M potassium phosphate (pH 7.4) and broken by sonication or passed through a French pressure cell at 1000 psi; the volume is adjusted to 140 ml with the same buffer. Step 2: Cell Supernatant. The cell supernatant is obtained by centrifugation at 13,500 gay for 2 hr. This treatment maximizes the recovery of ribosomal-bound enzyme with removal of membranes and cell debris. The supernatant is adjusted to 0.2% in Triton X-100 and 10% in glycerol. Step 3: Phosphocellulose Column. The following chromatography is carried out at room temperature. A 5 × 6 cm Whatman (Clifton, N J) Pl 1 phosphocellulose column is activated by washing with acid and base (see Kurland et al.14), equilibrated with 50 mM potassium phosphate (pH 7.4) containing 0.1% Triton X-100, 10% glycerol, and 1 mg/ml BSA, and finally washed with several column volumes of the same buffer lacking albumin; the albumin increases the yield of enzyme by blocking tightbinding sites on the column. The cell supernatant is applied to the column followed by washes of 300 ml of buffer A [0.1 M potassium phosphate (pH 7.4), 1% Triton X-100, 10% glycerol, and 0.5 mM dithiothreitol] containing 0.65 M NaCl and 200 ml of buffer B (buffer A at 0.1% Triton X-100 and containing 0.5 M NaC1). The enzyme is eluted from the column at 200 ml/ hr using 180 ml of buffer B containing 0.4 mM CDPdiacylglycerol followed by 100 ml of buffer B. At this point the enzyme, in a volume of about 140 ml, is essentially homogeneous as judged by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Step 4: DEAE-Sephadex Column. The pooled fractions from the P-11 column are dialyzed overnight against 800 ml of buffer C [20 mM potassium phosphate (pH 7.0), 0.2% Triton X-100, 10% glycerol, and 0.5 mM dithiothreitol]. The dialyzed enzyme is applied at 250 ml/hr to a 2.5 × 21 cm DEAE-Sephadex column equilibrated with buffer C. The column is washed with 200 ml of buffer C containing 0.1 M NaC1 followed by elution of the enzyme at 60 ml/hr with buffer C containing 1.2 M NaCl. The pooled peak of activity (10-15 ml) is dialyzed against buffer A for storage at - 80°. Purity. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation after Step 4 is over 95% homogeneous and has the same mobility (Mr 54,000) and specific activity as the enzyme prepared from cells lacking the overproducing plasmid. 6,12The preparation 14 C. G. Kudand, S. J. S. Hardy, and G. Mora, this series, Vol. 20, p. 381.
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PHOSPHATIDYLSERINE SYNTHASEFROME. coli
291
still contains significant amounts of CDPdiacylglycerol which binds to the DEAE-Sephadex column and elutes with the enzyme; with time, at 4 ° or above, this lipid is converted by the enzyme to phosphatidic acid and phosphatidylglycerol (see below). The bulk of the lipid can be removed by sedimentation of the enzyme through a 5 to 20% (w/v) glycerol gradient at 200,000 g~v for 12 hr in the presence of 0.1 M potassium phosphate (pH 7.4) as described by Carman and Dowhang; Triton X-100 (0.2%) or octylglucoside (30 mM) should be added back to the pooled peak of enzyme for storage. Properties
Stability. The enzyme is stable for several years at - 8 0 ° and several months at 4 ° when stored in buffer A. At ionic strengths below 0.1 the enzyme tends to precipitate, with irreversible loss of enzymatic activity. Although the enzyme is not isolated as a membrane protein, it does have a low affinity for nonionic detergent micelles (see below), and the presence of nonionic detergents (Triton X-100 or octylglucoside) above their critical micelle concentrations increases the stability of the enzyme. In the absence of detergent and in the presence of 0.1 M potassium phosphate buffer (pH 7.4), the enzyme is stable for several days at 4 °. The enzyme tends to adhere to negatively charged surfaces, and thus dilute solutions stored in glass containers lose activity rapidly; plastic containers are used when working with dilute solutions of the enzyme .6 There are no known specific inhibitors of the enzyme. Activity and Detergent Dependence. The enzyme is dependent on nonionic detergent for activity and is sensitive to the ratio of detergent (above its critical micelle concentration) to lipid substrate. 9 At 0.1 mM CDPdiacylglycerol the optimum activity occurs at a Triton to substrate molar ratio of 8 : 1. Activity is lower at either higher or lower ratios; the same is true at other fixed concentrations of substrate. At an 8 : 1 molar ratio the lipid substrate is completely dispersed in mixed micelles with the detergent, whereas below this ratio the substrate may exist partially in structures which are not catalytically competent or the higher surface concentration of substrate in the micelles may be inhibitory. The apparent reduction in Vmax at higher ratios appears to be due to dilution of the substrate within the mixed micelle surface, which reduces the affinity of the enzyme for the micelle (see below). As shown in Fig. 1A, the enzyme follows normal Michaelis-Menten kinetics if the activity is plotted as a function of the total concentration of detergent plus lipid substrate at fixed molar ratios of detergent to substrate. In such experiments the apparent Vmaxapproaches the true Vmaxas the ratio
292
SYNTHASES I
I
g
A
l
[34]
I
I
0.07'
I
I
/
0.05' 1 --g-
[]
D
-1.2
-0,8
-0,4
0
0.4
~ I
I
g
|
i
I
I
I
0.8
1.2
1.6
2.0
,1
mM I
g
l
I
i
B 0.04-
-
'
-16
-12
-8
-4
Vtmm
0
4
8
12
16
20
PL FIG. 1. Activity of phosphatidylserine synthase toward CDPdiacylglycerol (PL) in mixed micelles with Triton X-100 (TX). (A) The reciprocal of the velocity of the reaction (p.mol/ rain/rag) is plotted as a function of the reciprocal of the total concentration of PL and TX at varying molar ratios of TX : PL: [~, 8 : 1; *, 12 : 1; O, 16 : 1; A, 20 : 1. (B) Replot of the I / V intercepts (O) and the slopes ([~) from (A) as a function of the mole fraction of phospholipid in the mixed micelles can be used to determine the functions shown in the figure and summarized in Table I. [Data from G. M. Carman and W. Dowhan, J. Biol. Chem. 254, 8391 (1979).]
[34]
293
PHOSPHATIDYLSERINE SYNTHASEFROME. coli TABLE II KINETIC CONSTANTS FOR PHOSPHATIDYLSERINE SYNTHASEa Vmx 0zmol/min/mg)
nK~/x
xK~
Substrate
(mM)
(mole fraction)
CDP- 1,2-dipalmitoyl-L-glycerol CDPdiacyl-L-glycerol CDP- 1,2-dipalmitoyl-nL-glycerolb CDP- 1,2-dipalmitoyl-DL-glycerolc CDP- 1,2-dicaproyl-DL-glycerol b CDP- 1,2-dicaproyl-nL-glycerol c
71 79 50 50 90 90
1.4 1.2 3.4 3.2 3.3 3.3
0.065 O.064 0.10 0.058 0.06 0.03
From G. M. Carman and W. Dowhan, J. Biol. Chem. 254, 8391 (1979). b Concentration of both stereoisomers considered in the phospholipid term. c Only the L isomer considered in the phospholipid term and the o isomer considered in the inert Triton X-100 surface.
is increased. Replotting of the data (Fig. 1B) leads to the determination of three kinetic constants (see Warner and Dennis 15 for a discussion) at saturating concentrations of L-serine (I mM): the true Vm~x of 80 Ixmol/ min/mg; K A (total concentration of detergent plus substrate), which is a measure of the affinity of the enzyme for the mixed micelle surface; and the binding constant, Kam, for the substrate on the micelle surface. The values 15 for x and n were assumed to remain constant and represent the area of a substrate or a Triton molecule on the micelle surface and the area of the enzyme binding site on the micelle surface, respectively. Using such an analysis it is possible to assess the specificity for a number of lipid substrates (Table II). The Vm~x, K~, and KBm values are independent of fatty acid composition, and the enzyme is specific for the naturally occurring isomer of the phosphatidyl moiety. The D isomer is neither a substrate nor a competitive inhibitor but appears to be recognized as part of the inert surface of the micelle (no effect on Kam). Monoacylated lipids are neither substrates nor inhibitors of the enzyme. Reaction Mechanism. Analysis of the steric course of replacement of CMP by serine has established that the enzyme catalyzes synthesis of phosphatidylserine with retention of configuration at the phosphorus of the phosphatidyl moiety, 16 consistent with a two-step Ping-Pong reaction mechanism involving a covalently bound enzyme-phosphatidyl intermediate. The enzyme catalyzes the following reactions, 6A7which is also consis15 T. G. Warner and E. A. Dennis, J. Biol. Chem. 250, 8004 (1975). 16 C. R. H. Raetz, G. M. Carman, W. Dowhan, R.-T. Jiang, W. Waszkuc, W. Loffredo, and M.-D. Tsai, Biochemistry 26, 4022 (1987). 17 C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 249, 5038 (1974).
294
SYNTHASES
[34]
tent with this conclusion: the exchange between serine and phosphatidylserine and between CMP and CDPdiacylglycerol without the presence of other substrates or products; the hydrolysis of either phosphatidylserine or CDPdiacylglycerol to form phosphatidic acid; and the ability of glycerol (1 M) or sn-glycero-3-phosphate (0.8 mM) to act as a phosphatidyl acceptor in the presence of a large excess of enzyme. Physical Properties. The enzyme is unique among phospholipid biosynthetic enzymes of E. coli in that it is not membrane associated in cell-free extracts but is tightly bound to ribosomes. 2'18The association is primarily ionic since it can be disrupted by buffers with an ionic strength of 1.0 or greater. The polyamine spermidine in the physiological range (2-10 mM) also dissociates the enzyme from ribosomes. Dissociation is not brought about by nonionic detergents or the normal ionic strength of the assay conditions, but addition of either lipid substrate or lipid product under the assay conditions effects complete dissociation. These results suggest that the enzyme is associated with ribosomes primarily through ionic components of its active site and explains its high affinity for negatively charged surfaces. The effect of nonionic detergent, ionic strength, and CDPdiacylglycerol on the physical state of the enzyme has been investigated using changes in the sedimentation properties of the enzyme. 9 At the ionic strength of the normal assay mixture (0.6) and in the absence of detergent and lipid substrate, the enzyme sediments as a large monodisperse molecule with an M r of 500,000. Addition of Triton X-100 above its critical micelle concentration and in large excess over enzyme results in a polydisperse sedimentation pattern (Mr from 100,000 to 500,000) for the enzyme, consistent with weak interaction of the enzyme with the detergent micelles. Addition of increasing concentrations of CDPdiacylglycerol in the presence of a fixed Triton X-100 concentration (1.6 mM) results in a progressive increase in a monodisperse species with an Mr of 100,000. Optimal formation of the latter species occurs at a molar ratio of detergent to substrate of 8 : 1 (optimal for assay). Increasing the substrate concentration further but at a molar ratio less than 8 : 1 results in a polydisperse pattern for the enzyme, consistent with the substrate dilution within the micelle surface. A similar effect of ionic strength on sedimentation pattern of the enzyme, in the presence of detergent and substrate, is also seen which parallels the effect on enzymatic activity; that is, the species with an M r of 100,000 is favored as the optimal ionic strength for assay is approached. Comparison of the sedimentation properties of the various forms of the enzyme in glycerol gradients made in either D20 or H20 indicates that the species 18 K. Louie and W. Dowhan, J. Biol. Chem. 255, 1124 (1980).
[34]
PHOSPHATIDYLSERINE SYNTHASEFROME. coli
295
with an Mr of 100,000 has a density less than that of protein, consistent with it being a complex of protein and detergent-lipid mixed micelle, whereas the species with an Mr of 500,000 has the density of protein) 9 Although the multimeric structure of the enzyme cannot be determined from the above results, the species with an M r of 100,000 must be some multiple of the 54,000 subunit of the enzyme associated with a detergent-lipid substrate mixed micelle. These physical properties of the enzyme coupled with the kinetic analysis of the enzyme support a model in which catalysis occurs at the surface of a hydrophobic-hydrophilic interface. The affinity of the enzyme for such a surface is dependent on the presence and concentration of the negatively charged lipid substrate on that surface. Therefore, the two binding constants kinetically determined for the enzyme 9 appear to have some validity in physical terms since the enzyme does show low affinity for a micelle surface (KO), but the presence of the lipid substrate in that surface (Kam) results in very tight binding to the micelle; dilution of the lipid substrate within the surface decreases both surface affinity and enzymatic activity. The affinity of the enzyme for detergent micelles supplemented with the lipid substrate also extends to membranes of E. coli supplemented with various phospholipids. As noted above, the enzyme preferentially associates with the ribosomal fraction rather than the membrane fraction of cell lysates. However, both enzyme synthesized in vitro or the enzyme naturally present in cell lysates can be induced to associate preferentially with E. coli membranes which have been enriched in either CDPdiacylglycerol or phosphatidylserine2°; further enrichment with phosphatidylethanolamine does not induce membrane association, but the acidic phospholipids phosphatidylglycerol and cardiolipin do induce membrane association to some extent. The membrane-associated enzyme is kinetically competent, as evidenced by the formation of phosphatidylserine when serine is added to CDPdiacylglycerol-supplemented membranes. These results further support the above model for the action of this enzyme both in vivo and in vitro. S u b u n i t S t r u c t u r e . Based on the complete DNA sequence of the p s s
gene and verification by partial sequencing of the isolated protein, the enzyme is composed of a single monomer of 452 amino acids (Fig. 2) and has a molecular mass of 52,817 Da, 21which is consistent with the minimum size of the gene product expressed in vitro 2° and the mobility of the protein 19W. Dowhan, unpublished observation (1982). 20K. Louie, Y.-C. Chen, and W. Dowhan, J. Bacteriol. 165, 805 (1986). 21A. DeChavigny, P. N. Heacock, and W. Dowhan, J. Biol. Chem. 266, 5323 (1991).
296
SYNTHASES MLSKFKRNKH LEKIASAKQR ELDVRVLVDW DVPVYGVPIN LHQHDNIAYD LDDVNRPKSP VTPLVGLGKS LVRNIIQFVR LPYLYEINLR GMWVDDKWML QREKELELIR RIRIDRLISR
QQHLAQLPKI ICIVALYLEQ HRAQRGRIGA TREALGVLHF RYHLIRNRKM EIKNDIRLFR SLLNKTIFHL EGKKVEIIVG RFLSRLQYYV ITGNNLNPRA EHTTIVKHYR IL
SQSVDDVDFF DDGGKGILNA AASNTNADWY KGFIIDDSVL SDIMFEWVTQ QELRDAAYHF MPCAEQKLTI DKTANDFYIS NTDQLVVRLW WRLDLENAIL DLQSIADYPV
[34] YRPADFRETL LYEAKRQDDP CRMAQENPGV YSGASLNDVY NIMNGRGVNR QGDADNDQLS CTPYFNLPAI EDEPFKIIGA KDDDNTYHLK IHDPQLELAP KVRKLIRRLR
40 80 120 160 200 240 280 320 360 400 440 452
FIG. 2. Amino acid sequence for the phosphatidylserine synthase from E. coil as predicted from the DNA sequence of the pss gene [summarized from A. DeChavigny, P. N. Heacock, and W. Dowhan, J. Biol. Chem. 266, 5323 (1991)].
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis6; as noted above the native enzyme is some unknown multimer of this subunit. The amino-terminal methionine is not blocked. The predicted amino acid composition (Table III) is not unusual, and a hydrophobicity analysis of the linear sequence reveals two hydrophobic domains (residues 120-155 and 240-285) which may be involved in either substrate interaction or membrane association. The majority of the amino acid sequence is hydrophilic in nature and is characterized by an enrichment in basic amino acids at both the amino (5 of 9 amino acids) and carboxyl (10 of 22 amino acids) termini. These highly basic domains may be involved in association of the enzyme with negatively charged surfaces and the negatively charged lipid substrates. Properties of Mutants. Several mutants in the pss gene (mapping at 56 min of the E. coli chromosome) have been isolated which result in a conditional lethal, temperature-sensitive growth phenotype, n-24 These mutant strains stop growing after 4 to 5 generations at the restrictive temperature (42°) at which point the level of phosphatidylethanolamine declines from its normal level of 75% of the total phospholipid to 35%; phosphatidylserine does not accumulate owing to the activity of phosphatidylserine decarboxylase. The enzyme isolated from such mutants is also temperature-labile, consistent with the growth phenotype. Supplementation of the growth medium with 20 mM MgC12 suppresses the temperature22 A. Ohta and I. Shibuya, J. Bacteriol. 132, 434 (1977). 23 C. R. H. Raetz, G. A. Kantor, M. Nishijima, and K. F. Newman, J. Bacteriol. 139, 544 (1979). 24 I. Shibuya, C. Miyazaki, and A. Ohta, J. Bacteriol. 161, 1086 (1985).
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297
TABLE III PREDICTED AMINO ACID COMPOSITION OF PHOSPHATIDYLSERINE SYNTHASEa Amino acid
Residues per 52,800 Da
Asp Asn Thr Ser Glu
39 26 14 17 21 21 17 20 27 4 30
Gin
Pro Gly Ala Cys Val
Met Ile Leu Tyr Phe His Lys Trp Arg
8 37 51 19 15 13 26 7 40
a Summarized from A. DeChavigny, P. N. Heacock, and W. Dowhan, J. Biol. Chem. 266, 5323 0991)
sensitive growth phenotype of the mutants without returning the level of phosphatidylethanolamine to wild-type levels. Complete inactivation of the pss gene by insertion of a drug marker (pss :: kan) has shown that the residual phosphatidylethanolamine present in the above mutants results from the residual activity of the temperaturesensitive pss gene product. 21 Strains carrying the pss :: kan allele also stop growing when phosphatidylethanolamine levels reach 35%, but in the presence of millimolar concentrations of Ca 2÷ > Mg 2÷ > Sr 2÷ the strains grow near normally (in the order of effectiveness indicated) in rich medium. In contrast to bacteria carrying the temperature-sensitive point mutations grown in the presence of Mg 2÷ , the level of phosphatidylethanolamine is less than 0.01% of the total phospholipid of the cell. The ratio of phospholipid (which is now almost exclusively phosphatidylglycerol and cardiolipin) to membrane protein and the phospholipid fatty acid composition are nearly identical to those of wild-type cells. Therefore,
298
SYNTHASES
[35]
although phosphatidylethanolamine is an essential phospholipid under normal laboratory growth conditions, the nonspecific nature of the substitution by several divalent metal ions for this major membrane phospholipid suggests a primarily structural role for phosphatidylethanolamine. One possibility may be its role as an inert membrane matrix since this phospholipid carries no net charge; divalent metal ions could be acting to neutralize the high negative charge density of the remaining phospholipids. A second possibility may be the need for a phospholipid which can form nonbilayer structures possibly necessary in such processes as membrane fusion and translocation of macromolecules across membranes. This property of phosphatidylethanolamine 25could be substituted for by cardiolipin, which is known to display nonbilayer structures 26 in the presence of divalent metal ions with the same order of effectiveness as the three divalent metal ions which suppress the growth phenotype of the pss :: kan allele. 25 p. R. Cullis and B. de Kruijff, Biochim. Biophys. Acta 513, 31 (1978). 26 I. Vasilenko, B. de Kruijff, and A. J. Verkleij, Biochim. Biophys. Acta 684, 282 (1982).
[35] P h o s p h a t i d y l s e r i n e S y n t h a s e f r o m Y e a s t By GEORGE M. CARMAN and MYONGSUK BAE-LEE
Introduction Phosphatidylserine synthase (CDPdiacylglycerol-L-serine O-phosphatidyltransferase, EC 2.7.8.8) catalyzes the incorporation of serine into CDPdiacylglycerol + serine ~ phosphatidylserine + C M P
phosphatidylserine.l The enzyme plays an important role in the regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. 2 Phosphatidylserine synthase activity is associated with the mitochondrial and microsomal fractions of S. cerevisiae) '4 Phosphatidylserine synthase expression is regulated by inositol alone and in concert with serine, etha-
I j. N. Kanfer and E. P. K e n n e d y , J. Biol. Chem. 239, 1720 (1964). 2 G. M. C a r m a n and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 3 G. S. Cobon, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144, 265 (1974). 4 K. Kuchler, G. D a u m , a n d F. Paltauf, J. Bacteriol. 16S, 901 (1986).
METHODS IN ENZYMOLOGY.VOL. 209
Copyright © 1992by AcademicPress. Inc. All rights of reproduction in any form reserved.
PHOSPHATIDYLSERINE SYNTHASEFROME. coli
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[34] P h o s p h a t i d y l s e r i n e S y n t h a s e f r o m E s c h e r i c h i a coli By WILLIAM DOWHAN
Introduction (d)CDPdiacylglycerol + L-serine---> phosphatidylserine + (d)CMP
Phosphatidylserine synthases (EC 2.7.8.8, CDPdiacylglycerol-L-serine O-phosphatidyltransferase) from several gram-negative bacteria have been partially characterized and seem to be very similar in their physical and catalytic propertiesl'2; the enzyme from Escherichia coli has been extensively studied and characterized. Phospholipid biosynthetic enzymes in bacteria are generally found associated with membranes, but the phosphatidylserine synthases from gram-negative bacteria are found associated with the ribosomal fraction of cell lysates.l'2 The same reaction is catalyzed by the phosphatidylserine synthases from Saccharomyces cerevisiae 3 and the gram-positive bacilli, 4'5 but the enzymes from these organisms, unlike the enzymes from gram-negative bacteria, are dependent on added divalent metal ions for activity and are membrane associated. A similar CDPdiacylglycerol-dependent phosphatidylserine synthase activity has never been found in higher eukaryotic cells. Assay Method The enzyme is routinely assayed by following the incorporation of radiolabeled L-serine into chloroform-soluble material in the presence of a nonionic detergent such as Triton X-100; the ribose and deoxyribose forms of the liponucleotide are equivalent substrates. 6 In crude extracts either [3H]serine or [3-14C]serine should be used since the product is rapidly convened to phosphatidylethanolamine by the presence of the phosphatidylserine decarboxylase7; alternatively [U-14C]serine can be used if the decarboxylase is inhibited by inclusion of 10 mM hydroxylamine in the assay. Highly purified preparations of the enzyme can be assayed I A. Dutt and W. Dowhan, J. Bacteriol. 132, 159 (1977). 2 C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 247, 2008 (1972). 3 G. M. Carman and M. Bae-Lee, this volume [35]. 4 A. Dutt and W. Dowhan, J. Bacteriol. 147, 535 (1981). A. Dutt and W. Dowhan, Biochemistry 24, 1073 (1985). 6 T. J. Larson and W. Dowhan, Biochemistry 15, 5212 (1976). 7 W. Dowhan, W. T. Wickner, and E. P. Kennedy, J. Biol. Chem. 249, 3079 (1974).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SYNTHASES
[34]
spectrophotometrically by coupling the continuous release of CMP to the oxidation of NADH in the presence of ATP and phosphoenolpyruvate via the sequential actions of CMP kinase, pyruvate kinase, and lactate dehydrogenase.8'9 P r o c e d u r e . 6 If necessary the enzyme is diluted with 0.1 M potassium phosphate buffer (pH 7.4) containing 0.1% (1.6 mM) Triton X-100 and 1 mg/ml of bovine serum albumin (BSA). Dilution and assay are done in polypropylene tubes since the dilute, purified enzyme tends to adhere to glass; serum albumin and high protein concentrations minimize this problem. The final concentrations of the components of the assay mixture are 0.67 mM CDPdiacylglycerol (synthesized from egg yolk phosphatidylcholine as described elsewhere in this volumel°), 0.5 mM L-serine (radiolabeled as desired to a specific activity of 2-4/zCi//zmol), 0.1% Triton X-100, 0.1 M potassium phosphate buffer (pH 7.4), and 1 mg/ml BSA; an ionic strength of 0.3 or higher is optimal. 9 The reaction is initiated by adding 10/zl of an enzyme solution to 50/zl of a 1.2-fold concentrated stock solution of the above assay mixture. The reaction mixture is incubated in a 12-ml polypropylene tube at 30° for 10 min. The reaction is stopped by adding, in the following order, 0.5 ml of methanol (0.1 N in HC1), 1.5 ml chloroform, and 3.0 ml of 1 M MgCI2. After thorough mixing of the two phases and separation of the phases by centrifugation at 1000 gav in a clinical centrifuge at room temperature, 1 ml of the chloroform phase (lower phase) is removed, evaporated to dryness at 65°, and counted for radioactivity using any commercially available scintillation fluid. One unit of activity is defined as the amount of enzyme which converts 1 /zmol of L-serine into chloroform-soluble material in 1 min under the above conditions. The assay is linear for the pure enzyme up to about 50% conversion of the limiting substrate. When CDPdiacylglycerol is the limiting substrate, total conversion approaches only 95%, probably because of an inherent hydrolase activity which is 1% of the synthase rate. In crude preparations the linearity may fall off before reaching 50% conversion depending on the degree of contamination by the endogenous, membrane-associated CDP-diacylglycerol hydrolase activityll; this activity can be inhibited by 1 mM ATP.
8 G. M. Carman and W. Dowhan, J. Lipid Res. 19, 519 (1978). 9 G. M. Carman and W. Dowhan, J. Biol. Chem. 2,54, 8391 (1979). 10 G. M. Carman and A. S. Fischl, this volume [36]. u C. R. H. Raetz, C. B. Hirschberg, W. Dowhan, W. T. Wickner, and E. P. Kennedy, J. Biol. Chem.247, 2245 (1972).
[34]
PHOSPHATIDYLSERINE SYNTHASEFROME. coli
289
TABLE I PURIFICATION OF PHOSPHATIDYLSERINE SYNTHASEa
Step I. 2. 3. 4.
Cell-free extract Cell supernatant Phosphocellulose DEAE-Sephadex
Total protein
Specific activity (units/mg)
Yield (%)
3.1 g 3.0 g ND b 34 mg
0.80 0.87 ND 39
100 104 70 54
a Starting with 20 g of cell paste from E. coli strain JA200/pPS3155-k as described by A. Ohta, K. Waggoner, K. Louie, and W. Dowhan, J. Biol. Chem. 256, 219 (1981). b Not determined.
Purification Procedure It is possible to obtain near gram quantities of highly purified enzyme by utilizing a purification scheme which relies on specific elution of the enzyme from phosphocellulose using the liponucleotide substrate 6 and starting with a strain of E. coli containing a plasmid-borne copy of the structural gene (pss) which directs the overproduction of the enzyme 100to 200-fold. 12The structural gene for the phosphatidylserine synthase has been introduced into a derivative of plasmid pBR322 (pPS3155-h) which carries the N O P region of h phage13; the N O P region contains the h origin of replication, a temperature-sensitive ~ repressor, and the essential promoters. At 30° this plasmid replicates from the normal pBR322 origin and is carried at about 11 copies per cell. Induction at 42° results in inactivation of the h repressor and an increase in the copy number of the plasmid to greater than 100 per cell. Because the production of the pss gene product is gene dose-dependent, cells carrying this plasmid grown at 42 ° have greatly elevated levels of phosphatidylserine synthase. Except where indicated the following steps (taken from Ohta et a l ) 2 and summarized in Table I) are carried out at 4 °. Growth of Cells. Cells can be grown in batches of 1 liter or less in shaking flasks or in 100-liter amounts in a fermentor using SB broth [12 g/ liter of Bacto-tryptone (Difco, Detroit, MI), 25 g/liter of yeast extract, 0.1 M potassium phosphate (pH 7.0), and 0.5% glucose] supplemented with 50/zg/ml ampicillin (the plasmid drug marker). Strain JA200/pPS3155-)t is grown to mid-log phase (OD550 1.5) at 30°, rapidly shifted to 42° for 30 min, and then incubated at 37° for 4 hr before harvesting the cells; high aeration and rapid shaking are maintained throughout the growth period. The yield 12 A. Ohta, K. Waggoner, K. Louie, and W. Dowhan, J. Biol. Chem. 256, 219 (1981). t3 R. N. Rao and S. G. Rogers, Gene 3, 247 0978).
290
SYNTHASES
[34]
of cells is about 12 g/liter of wet weight cells, with a specific activity for the phosphatidylserine synthase ranging between 100- and 200-fold over that from strain JA200 lacking the plasmid. The cell paste can be stored at - 8 0 ° for years. Step 1: Cell-Free Extract. Cell paste (20 g) of strain JA200/pPS3155-~ is suspended in 70 ml of buffer containing 0.1 M potassium phosphate (pH 7.4) and broken by sonication or passed through a French pressure cell at 1000 psi; the volume is adjusted to 140 ml with the same buffer. Step 2: Cell Supernatant. The cell supernatant is obtained by centrifugation at 13,500 gay for 2 hr. This treatment maximizes the recovery of ribosomal-bound enzyme with removal of membranes and cell debris. The supernatant is adjusted to 0.2% in Triton X-100 and 10% in glycerol. Step 3: Phosphocellulose Column. The following chromatography is carried out at room temperature. A 5 × 6 cm Whatman (Clifton, N J) Pl 1 phosphocellulose column is activated by washing with acid and base (see Kurland et al.14), equilibrated with 50 mM potassium phosphate (pH 7.4) containing 0.1% Triton X-100, 10% glycerol, and 1 mg/ml BSA, and finally washed with several column volumes of the same buffer lacking albumin; the albumin increases the yield of enzyme by blocking tightbinding sites on the column. The cell supernatant is applied to the column followed by washes of 300 ml of buffer A [0.1 M potassium phosphate (pH 7.4), 1% Triton X-100, 10% glycerol, and 0.5 mM dithiothreitol] containing 0.65 M NaCl and 200 ml of buffer B (buffer A at 0.1% Triton X-100 and containing 0.5 M NaC1). The enzyme is eluted from the column at 200 ml/ hr using 180 ml of buffer B containing 0.4 mM CDPdiacylglycerol followed by 100 ml of buffer B. At this point the enzyme, in a volume of about 140 ml, is essentially homogeneous as judged by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Step 4: DEAE-Sephadex Column. The pooled fractions from the P-11 column are dialyzed overnight against 800 ml of buffer C [20 mM potassium phosphate (pH 7.0), 0.2% Triton X-100, 10% glycerol, and 0.5 mM dithiothreitol]. The dialyzed enzyme is applied at 250 ml/hr to a 2.5 × 21 cm DEAE-Sephadex column equilibrated with buffer C. The column is washed with 200 ml of buffer C containing 0.1 M NaC1 followed by elution of the enzyme at 60 ml/hr with buffer C containing 1.2 M NaCl. The pooled peak of activity (10-15 ml) is dialyzed against buffer A for storage at - 80°. Purity. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation after Step 4 is over 95% homogeneous and has the same mobility (Mr 54,000) and specific activity as the enzyme prepared from cells lacking the overproducing plasmid. 6,12The preparation 14 C. G. Kudand, S. J. S. Hardy, and G. Mora, this series, Vol. 20, p. 381.
[34]
PHOSPHATIDYLSERINE SYNTHASEFROME. coli
291
still contains significant amounts of CDPdiacylglycerol which binds to the DEAE-Sephadex column and elutes with the enzyme; with time, at 4 ° or above, this lipid is converted by the enzyme to phosphatidic acid and phosphatidylglycerol (see below). The bulk of the lipid can be removed by sedimentation of the enzyme through a 5 to 20% (w/v) glycerol gradient at 200,000 g~v for 12 hr in the presence of 0.1 M potassium phosphate (pH 7.4) as described by Carman and Dowhang; Triton X-100 (0.2%) or octylglucoside (30 mM) should be added back to the pooled peak of enzyme for storage. Properties
Stability. The enzyme is stable for several years at - 8 0 ° and several months at 4 ° when stored in buffer A. At ionic strengths below 0.1 the enzyme tends to precipitate, with irreversible loss of enzymatic activity. Although the enzyme is not isolated as a membrane protein, it does have a low affinity for nonionic detergent micelles (see below), and the presence of nonionic detergents (Triton X-100 or octylglucoside) above their critical micelle concentrations increases the stability of the enzyme. In the absence of detergent and in the presence of 0.1 M potassium phosphate buffer (pH 7.4), the enzyme is stable for several days at 4 °. The enzyme tends to adhere to negatively charged surfaces, and thus dilute solutions stored in glass containers lose activity rapidly; plastic containers are used when working with dilute solutions of the enzyme .6 There are no known specific inhibitors of the enzyme. Activity and Detergent Dependence. The enzyme is dependent on nonionic detergent for activity and is sensitive to the ratio of detergent (above its critical micelle concentration) to lipid substrate. 9 At 0.1 mM CDPdiacylglycerol the optimum activity occurs at a Triton to substrate molar ratio of 8 : 1. Activity is lower at either higher or lower ratios; the same is true at other fixed concentrations of substrate. At an 8 : 1 molar ratio the lipid substrate is completely dispersed in mixed micelles with the detergent, whereas below this ratio the substrate may exist partially in structures which are not catalytically competent or the higher surface concentration of substrate in the micelles may be inhibitory. The apparent reduction in Vmax at higher ratios appears to be due to dilution of the substrate within the mixed micelle surface, which reduces the affinity of the enzyme for the micelle (see below). As shown in Fig. 1A, the enzyme follows normal Michaelis-Menten kinetics if the activity is plotted as a function of the total concentration of detergent plus lipid substrate at fixed molar ratios of detergent to substrate. In such experiments the apparent Vmaxapproaches the true Vmaxas the ratio
292
SYNTHASES I
I
g
A
l
[34]
I
I
0.07'
I
I
/
0.05' 1 --g-
[]
D
-1.2
-0,8
-0,4
0
0.4
~ I
I
g
|
i
I
I
I
0.8
1.2
1.6
2.0
,1
mM I
g
l
I
i
B 0.04-
-
'
-16
-12
-8
-4
Vtmm
0
4
8
12
16
20
PL FIG. 1. Activity of phosphatidylserine synthase toward CDPdiacylglycerol (PL) in mixed micelles with Triton X-100 (TX). (A) The reciprocal of the velocity of the reaction (p.mol/ rain/rag) is plotted as a function of the reciprocal of the total concentration of PL and TX at varying molar ratios of TX : PL: [~, 8 : 1; *, 12 : 1; O, 16 : 1; A, 20 : 1. (B) Replot of the I / V intercepts (O) and the slopes ([~) from (A) as a function of the mole fraction of phospholipid in the mixed micelles can be used to determine the functions shown in the figure and summarized in Table I. [Data from G. M. Carman and W. Dowhan, J. Biol. Chem. 254, 8391 (1979).]
[34]
293
PHOSPHATIDYLSERINE SYNTHASEFROME. coli TABLE II KINETIC CONSTANTS FOR PHOSPHATIDYLSERINE SYNTHASEa Vmx 0zmol/min/mg)
nK~/x
xK~
Substrate
(mM)
(mole fraction)
CDP- 1,2-dipalmitoyl-L-glycerol CDPdiacyl-L-glycerol CDP- 1,2-dipalmitoyl-nL-glycerolb CDP- 1,2-dipalmitoyl-DL-glycerolc CDP- 1,2-dicaproyl-DL-glycerol b CDP- 1,2-dicaproyl-nL-glycerol c
71 79 50 50 90 90
1.4 1.2 3.4 3.2 3.3 3.3
0.065 O.064 0.10 0.058 0.06 0.03
From G. M. Carman and W. Dowhan, J. Biol. Chem. 254, 8391 (1979). b Concentration of both stereoisomers considered in the phospholipid term. c Only the L isomer considered in the phospholipid term and the o isomer considered in the inert Triton X-100 surface.
is increased. Replotting of the data (Fig. 1B) leads to the determination of three kinetic constants (see Warner and Dennis 15 for a discussion) at saturating concentrations of L-serine (I mM): the true Vm~x of 80 Ixmol/ min/mg; K A (total concentration of detergent plus substrate), which is a measure of the affinity of the enzyme for the mixed micelle surface; and the binding constant, Kam, for the substrate on the micelle surface. The values 15 for x and n were assumed to remain constant and represent the area of a substrate or a Triton molecule on the micelle surface and the area of the enzyme binding site on the micelle surface, respectively. Using such an analysis it is possible to assess the specificity for a number of lipid substrates (Table II). The Vm~x, K~, and KBm values are independent of fatty acid composition, and the enzyme is specific for the naturally occurring isomer of the phosphatidyl moiety. The D isomer is neither a substrate nor a competitive inhibitor but appears to be recognized as part of the inert surface of the micelle (no effect on Kam). Monoacylated lipids are neither substrates nor inhibitors of the enzyme. Reaction Mechanism. Analysis of the steric course of replacement of CMP by serine has established that the enzyme catalyzes synthesis of phosphatidylserine with retention of configuration at the phosphorus of the phosphatidyl moiety, 16 consistent with a two-step Ping-Pong reaction mechanism involving a covalently bound enzyme-phosphatidyl intermediate. The enzyme catalyzes the following reactions, 6A7which is also consis15 T. G. Warner and E. A. Dennis, J. Biol. Chem. 250, 8004 (1975). 16 C. R. H. Raetz, G. M. Carman, W. Dowhan, R.-T. Jiang, W. Waszkuc, W. Loffredo, and M.-D. Tsai, Biochemistry 26, 4022 (1987). 17 C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 249, 5038 (1974).
294
SYNTHASES
[34]
tent with this conclusion: the exchange between serine and phosphatidylserine and between CMP and CDPdiacylglycerol without the presence of other substrates or products; the hydrolysis of either phosphatidylserine or CDPdiacylglycerol to form phosphatidic acid; and the ability of glycerol (1 M) or sn-glycero-3-phosphate (0.8 mM) to act as a phosphatidyl acceptor in the presence of a large excess of enzyme. Physical Properties. The enzyme is unique among phospholipid biosynthetic enzymes of E. coli in that it is not membrane associated in cell-free extracts but is tightly bound to ribosomes. 2'18The association is primarily ionic since it can be disrupted by buffers with an ionic strength of 1.0 or greater. The polyamine spermidine in the physiological range (2-10 mM) also dissociates the enzyme from ribosomes. Dissociation is not brought about by nonionic detergents or the normal ionic strength of the assay conditions, but addition of either lipid substrate or lipid product under the assay conditions effects complete dissociation. These results suggest that the enzyme is associated with ribosomes primarily through ionic components of its active site and explains its high affinity for negatively charged surfaces. The effect of nonionic detergent, ionic strength, and CDPdiacylglycerol on the physical state of the enzyme has been investigated using changes in the sedimentation properties of the enzyme. 9 At the ionic strength of the normal assay mixture (0.6) and in the absence of detergent and lipid substrate, the enzyme sediments as a large monodisperse molecule with an M r of 500,000. Addition of Triton X-100 above its critical micelle concentration and in large excess over enzyme results in a polydisperse sedimentation pattern (Mr from 100,000 to 500,000) for the enzyme, consistent with weak interaction of the enzyme with the detergent micelles. Addition of increasing concentrations of CDPdiacylglycerol in the presence of a fixed Triton X-100 concentration (1.6 mM) results in a progressive increase in a monodisperse species with an Mr of 100,000. Optimal formation of the latter species occurs at a molar ratio of detergent to substrate of 8 : 1 (optimal for assay). Increasing the substrate concentration further but at a molar ratio less than 8 : 1 results in a polydisperse pattern for the enzyme, consistent with the substrate dilution within the micelle surface. A similar effect of ionic strength on sedimentation pattern of the enzyme, in the presence of detergent and substrate, is also seen which parallels the effect on enzymatic activity; that is, the species with an M r of 100,000 is favored as the optimal ionic strength for assay is approached. Comparison of the sedimentation properties of the various forms of the enzyme in glycerol gradients made in either D20 or H20 indicates that the species 18 K. Louie and W. Dowhan, J. Biol. Chem. 255, 1124 (1980).
[34]
PHOSPHATIDYLSERINE SYNTHASEFROME. coli
295
with an Mr of 100,000 has a density less than that of protein, consistent with it being a complex of protein and detergent-lipid mixed micelle, whereas the species with an Mr of 500,000 has the density of protein) 9 Although the multimeric structure of the enzyme cannot be determined from the above results, the species with an M r of 100,000 must be some multiple of the 54,000 subunit of the enzyme associated with a detergent-lipid substrate mixed micelle. These physical properties of the enzyme coupled with the kinetic analysis of the enzyme support a model in which catalysis occurs at the surface of a hydrophobic-hydrophilic interface. The affinity of the enzyme for such a surface is dependent on the presence and concentration of the negatively charged lipid substrate on that surface. Therefore, the two binding constants kinetically determined for the enzyme 9 appear to have some validity in physical terms since the enzyme does show low affinity for a micelle surface (KO), but the presence of the lipid substrate in that surface (Kam) results in very tight binding to the micelle; dilution of the lipid substrate within the surface decreases both surface affinity and enzymatic activity. The affinity of the enzyme for detergent micelles supplemented with the lipid substrate also extends to membranes of E. coli supplemented with various phospholipids. As noted above, the enzyme preferentially associates with the ribosomal fraction rather than the membrane fraction of cell lysates. However, both enzyme synthesized in vitro or the enzyme naturally present in cell lysates can be induced to associate preferentially with E. coli membranes which have been enriched in either CDPdiacylglycerol or phosphatidylserine2°; further enrichment with phosphatidylethanolamine does not induce membrane association, but the acidic phospholipids phosphatidylglycerol and cardiolipin do induce membrane association to some extent. The membrane-associated enzyme is kinetically competent, as evidenced by the formation of phosphatidylserine when serine is added to CDPdiacylglycerol-supplemented membranes. These results further support the above model for the action of this enzyme both in vivo and in vitro. S u b u n i t S t r u c t u r e . Based on the complete DNA sequence of the p s s
gene and verification by partial sequencing of the isolated protein, the enzyme is composed of a single monomer of 452 amino acids (Fig. 2) and has a molecular mass of 52,817 Da, 21which is consistent with the minimum size of the gene product expressed in vitro 2° and the mobility of the protein 19W. Dowhan, unpublished observation (1982). 20K. Louie, Y.-C. Chen, and W. Dowhan, J. Bacteriol. 165, 805 (1986). 21A. DeChavigny, P. N. Heacock, and W. Dowhan, J. Biol. Chem. 266, 5323 (1991).
296
SYNTHASES MLSKFKRNKH LEKIASAKQR ELDVRVLVDW DVPVYGVPIN LHQHDNIAYD LDDVNRPKSP VTPLVGLGKS LVRNIIQFVR LPYLYEINLR GMWVDDKWML QREKELELIR RIRIDRLISR
QQHLAQLPKI ICIVALYLEQ HRAQRGRIGA TREALGVLHF RYHLIRNRKM EIKNDIRLFR SLLNKTIFHL EGKKVEIIVG RFLSRLQYYV ITGNNLNPRA EHTTIVKHYR IL
SQSVDDVDFF DDGGKGILNA AASNTNADWY KGFIIDDSVL SDIMFEWVTQ QELRDAAYHF MPCAEQKLTI DKTANDFYIS NTDQLVVRLW WRLDLENAIL DLQSIADYPV
[34] YRPADFRETL LYEAKRQDDP CRMAQENPGV YSGASLNDVY NIMNGRGVNR QGDADNDQLS CTPYFNLPAI EDEPFKIIGA KDDDNTYHLK IHDPQLELAP KVRKLIRRLR
40 80 120 160 200 240 280 320 360 400 440 452
FIG. 2. Amino acid sequence for the phosphatidylserine synthase from E. coil as predicted from the DNA sequence of the pss gene [summarized from A. DeChavigny, P. N. Heacock, and W. Dowhan, J. Biol. Chem. 266, 5323 (1991)].
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis6; as noted above the native enzyme is some unknown multimer of this subunit. The amino-terminal methionine is not blocked. The predicted amino acid composition (Table III) is not unusual, and a hydrophobicity analysis of the linear sequence reveals two hydrophobic domains (residues 120-155 and 240-285) which may be involved in either substrate interaction or membrane association. The majority of the amino acid sequence is hydrophilic in nature and is characterized by an enrichment in basic amino acids at both the amino (5 of 9 amino acids) and carboxyl (10 of 22 amino acids) termini. These highly basic domains may be involved in association of the enzyme with negatively charged surfaces and the negatively charged lipid substrates. Properties of Mutants. Several mutants in the pss gene (mapping at 56 min of the E. coli chromosome) have been isolated which result in a conditional lethal, temperature-sensitive growth phenotype, n-24 These mutant strains stop growing after 4 to 5 generations at the restrictive temperature (42°) at which point the level of phosphatidylethanolamine declines from its normal level of 75% of the total phospholipid to 35%; phosphatidylserine does not accumulate owing to the activity of phosphatidylserine decarboxylase. The enzyme isolated from such mutants is also temperature-labile, consistent with the growth phenotype. Supplementation of the growth medium with 20 mM MgC12 suppresses the temperature22 A. Ohta and I. Shibuya, J. Bacteriol. 132, 434 (1977). 23 C. R. H. Raetz, G. A. Kantor, M. Nishijima, and K. F. Newman, J. Bacteriol. 139, 544 (1979). 24 I. Shibuya, C. Miyazaki, and A. Ohta, J. Bacteriol. 161, 1086 (1985).
[34]
PHOSPHATIDYLSERINE SYNTHASE FROM E. coli
297
TABLE III PREDICTED AMINO ACID COMPOSITION OF PHOSPHATIDYLSERINE SYNTHASEa Amino acid
Residues per 52,800 Da
Asp Asn Thr Ser Glu
39 26 14 17 21 21 17 20 27 4 30
Gin
Pro Gly Ala Cys Val
Met Ile Leu Tyr Phe His Lys Trp Arg
8 37 51 19 15 13 26 7 40
a Summarized from A. DeChavigny, P. N. Heacock, and W. Dowhan, J. Biol. Chem. 266, 5323 0991)
sensitive growth phenotype of the mutants without returning the level of phosphatidylethanolamine to wild-type levels. Complete inactivation of the pss gene by insertion of a drug marker (pss :: kan) has shown that the residual phosphatidylethanolamine present in the above mutants results from the residual activity of the temperaturesensitive pss gene product. 21 Strains carrying the pss :: kan allele also stop growing when phosphatidylethanolamine levels reach 35%, but in the presence of millimolar concentrations of Ca 2÷ > Mg 2÷ > Sr 2÷ the strains grow near normally (in the order of effectiveness indicated) in rich medium. In contrast to bacteria carrying the temperature-sensitive point mutations grown in the presence of Mg 2÷ , the level of phosphatidylethanolamine is less than 0.01% of the total phospholipid of the cell. The ratio of phospholipid (which is now almost exclusively phosphatidylglycerol and cardiolipin) to membrane protein and the phospholipid fatty acid composition are nearly identical to those of wild-type cells. Therefore,
298
SYNTHASES
[35]
although phosphatidylethanolamine is an essential phospholipid under normal laboratory growth conditions, the nonspecific nature of the substitution by several divalent metal ions for this major membrane phospholipid suggests a primarily structural role for phosphatidylethanolamine. One possibility may be its role as an inert membrane matrix since this phospholipid carries no net charge; divalent metal ions could be acting to neutralize the high negative charge density of the remaining phospholipids. A second possibility may be the need for a phospholipid which can form nonbilayer structures possibly necessary in such processes as membrane fusion and translocation of macromolecules across membranes. This property of phosphatidylethanolamine 25could be substituted for by cardiolipin, which is known to display nonbilayer structures 26 in the presence of divalent metal ions with the same order of effectiveness as the three divalent metal ions which suppress the growth phenotype of the pss :: kan allele. 25 p. R. Cullis and B. de Kruijff, Biochim. Biophys. Acta 513, 31 (1978). 26 I. Vasilenko, B. de Kruijff, and A. J. Verkleij, Biochim. Biophys. Acta 684, 282 (1982).
[35] P h o s p h a t i d y l s e r i n e S y n t h a s e f r o m Y e a s t By GEORGE M. CARMAN and MYONGSUK BAE-LEE
Introduction Phosphatidylserine synthase (CDPdiacylglycerol-L-serine O-phosphatidyltransferase, EC 2.7.8.8) catalyzes the incorporation of serine into CDPdiacylglycerol + serine ~ phosphatidylserine + C M P
phosphatidylserine.l The enzyme plays an important role in the regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. 2 Phosphatidylserine synthase activity is associated with the mitochondrial and microsomal fractions of S. cerevisiae) '4 Phosphatidylserine synthase expression is regulated by inositol alone and in concert with serine, etha-
I j. N. Kanfer and E. P. K e n n e d y , J. Biol. Chem. 239, 1720 (1964). 2 G. M. C a r m a n and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 3 G. S. Cobon, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144, 265 (1974). 4 K. Kuchler, G. D a u m , a n d F. Paltauf, J. Bacteriol. 16S, 901 (1986).
METHODS IN ENZYMOLOGY.VOL. 209
Copyright © 1992by AcademicPress. Inc. All rights of reproduction in any form reserved.
