Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol

Larson, Timothy J.; Dowhan, William

doi:10.1021/bi00669a003

Cited by 97 publications

(87 citation statements)

References 29 publications

(32 reference statements)

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“…This observation was supported by the fact that there was no detectable product overexpressed in the maxicells by using the native pss gene, as shown in the Results, which might be expected since the PSS protein could be constitutively produced in low amounts as found in E. coli (22).…”

Section: Discussionsupporting

confidence: 62%

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The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

Taylor

1997

J Bacteriol

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The Helicobacter pylori pss gene, coding for phosphatidylserine synthase (PSS), was cloned and sequenced in this study. A polypeptide of 237 amino acids was deduced from the PSS sequence. H. pylori PSS exhibits significant amino acid sequence identity with the PSS proteins found in the archaebacterium Methanococcus jannaschii, the gram-positive bacterium Bacillus subtilis, and the yeast Saccharomyces cerevisiae but none with its Escherichia coli counterpart. Expression of the putative pss gene in maxicells gave rise to a product of ϳ26 kDa, which is in agreement with the predicted molecular mass of 26,617 Da. A manganese-dependent PSS activity was found in the membrane fractions of the E. coli cells overexpressing the H. pylori pss gene product. This result indicates that this enzyme is a membrane-bound protein, a conclusion which is supported by the fact that the PSS protein contains several local hydrophobic segments which could form transmembrane helices. The pss gene was inactivated with a chloramphenicol acetyltransferase cassette on the plasmid. However, an isogenic pss gene-disrupted mutant of H. pylori UA802 could not be obtained, suggesting that this enzyme plays an essential role in the growth of this organism.

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Section: Discussionsupporting

confidence: 62%

“…The same mechanism was also proposed for S. cerevisiae PSS and E. coli phosphatidylglycerophosphate synthase (1,14). In contrast, the E. coli PSS reaction has characteristics of ping-pong mechanisms in which one or more products are released before all substrates have been added (22,34).…”

Section: Discussionmentioning

confidence: 68%

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

Taylor

1997

J Bacteriol

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“…The former possibility would be excluded if a null mutant can be constructed by deletion of the entire pgsA gene, as was done in the case of the cls gene (24), instead of an insertion into the gene, as was used in the present mutant. The latter side reaction might be catalyzed by phosphatidylserine synthase, since a substantially purified preparation of this enzyme was shown to catalyze, though very slowly, the formation of phosphatidylglycerol or phosphatidylglycerophosphate when the substrate serine was replaced by glycerol or sn-glycerol 3-phosphate, respectively (20). To suggest the side reaction more strongly, a further examination with a phosphatidylserine synthase preparation purified from a pgsA null mutant will be needed.…”

Section: Resultsmentioning

confidence: 99%

Viability of an Escherichia coli pgsA Null Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin

2000

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Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40°C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783-787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.Since the discovery of phosphatidylglycerol by Benson and Maruo in 1958 (3), the acidic phospholipid has been found in almost every organism and is believed to play essential roles in various cellular processes, such as SecA protein-dependent translocation of proteins across the membrane and rejuvenation of DnaA protein into an active ATP-bound form in the initiation of oriC DNA replication, based on results obtained mainly from in vitro studies (9, 32).The Escherichia coli pgsA3 allele encodes a defective phosphatidylglycerophosphate synthase with low activity, resulting in cells defective in the major acidic phospholipids, phosphatidylglycerol and cardiolipin (23). The pgsA mutation is lethal in wild-type cells, while the cls mutations, resulting in reduction of cardiolipin content, affect their growth only slightly (13,24). Therefore, the lethality and growth arrest phenotype of acidic phospholipid-deficiency caused by pgsA3 and reduced expression of pgsA, which is under the control of the lac promoter, are considered indications of the involvement of the acidic phospholipid in essential cellular functions (9,11,12,32). The lethality is suppressed by the lack of or by certain mutational changes in the major outer membrane lipoprotein (Braun's lipoprotein), which consumes equimolar amounts of phosphatidylglycerol for processing of prolipoprotein, the primary gene product of lpp (2, 31). There are two alternatives to explain the suppression: (i) a drain of the limited acidic phospholipid pool required for certain essential functions in pgsA mutants is relieved by a ...

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“…Phosphatidylserine synthase may be able to use a Ser-Glu dipeptide, perhaps the by-product of protein degradation, instead of serine. While it has been shown that the phosphatidylserine synthase of E. coli is very specifi c for serine, it does slowly transfer the CDP-DAG phosphatidyl moiety to glycerol, glycerol 3-P, and phosphatidylglycerol ( 38,39 ). Accordingly, purifi ed phosphatidylserine synthase needs to be tested to determine if PSE can be formed in vitro using CDP-DAG and serylglutamate as substrates ( 39 ).…”

Section: Discussionmentioning

confidence: 99%

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

Garrett

Raetz

Richardson

et al. 2009

Journal of Lipid Research

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dylglycerol phosphate ( 3 ). For each of these lipids, there can exist several molecular species arising from the different lengths, unsaturation, and/or cyclopropane analogs of the acyl chains ( 4 ). The complexity of the E. coli lipidome, however, is even greater than can be explained by acyl chain heterogeneity. Numerous additional minor lipids are present in wild-type cells, as judged by isotopic labeling experiments and two-dimensional TLC ( 3 ). Many of these species cannot be identifi ed by their migration with standards of known lipids or biosynthetic precursors.Electrospray ionization-mass spectrometry (ESI-MS) is well suited to the analysis of intact lipids ( 5 ). Fragmentation during ionization is minimized and the sensitivity is high ( 6, 7 ). In addition, collision-induced dissociation mass spectrometry (MS/MS) allows for the structural analysis of the lipid ion of interest and, when combined with the high mass accuracy of time-of-fl ight mass spectrometers, can be used to propose a molecular formula for a particular ion.Current applications of mass spectrometry to lipid analysis have focused mainly on the quantifi cation of known lipid species, often coupling liquid chromatography directly to the mass spectrometer ( 8-10 ). These analyses compare levels of known lipid species present under various growth conditions or disease states. However, important changes in levels of unknown or minor lipids are diffi cult to analyze without knowledge of their structures and the availability of appropriate standards. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) are the major membrane glycerophospholipids of Escherichia coli , constituting about 10% of the dry weight of the cell ( 1, 2 ). Important minor lipids include the precursors phosphatidic acid, CDP-diacylglycerol, phosphatidylserine, and phosphati-

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Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol

Cited by 97 publications

References 29 publications

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

Viability of an Escherichia coli pgsA Null Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

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