A second Escherichia coli protein with CL synthase activity.

The Escherichia coli open reading frame f413, which has the potential to code for a polypeptide homologous to cardiolipin (CL) synthase, has been cloned. Its polypeptide product has a molecular mass of 48 kDa, is membrane-bound, and catalyzes CL formation but does not hydrolyze CL. A comparison of the sequences predicted for the polypeptides encoded by f413 and cls indicates that the N-terminal residues specified by cls may be unnecessary for CL synthase activity. Construction of a truncated cls gene and characterization of its polypeptide product have confirmed this conclusion.

Cloning of the Bacillus firmus OF4 cls gene and characterization of its gene product.

The gene that codes for cardiolipin (CL) synthase and an adjacent gene that codes for a MecA homolog in the alkaliphilic bacteria Bacillus firmus OF4 have been cloned and sequenced (GenBank accession number U88888). The cls gene contains 1509 nucleotides, corresponding to a polypeptide of 57.9 kDa. The predicted amino acid sequence has 129 identities and 100 similarities with the Escherichia coli CL synthase. Homologies were also noted with polypeptide sequences from putative cls genes from Bacillus subtilis and Pseudomonas putida. Conserved histidine, tyrosine, and serine residues may be part of the active site and participate in phosphatidyl group transfer. The B. firmus OF4 cls gene product was inserted into plasmid pET3 to form a recombinant plasmid pDG2, which overproduces CL synthase in E. coli. A membrane fraction containing the overproduced enzyme converts phosphatidylglycerol to CL and glycerol. The B. firmus enzyme is stimulated by potassium phosphate, inhibited by CL and phosphatidate, and has a slightly higher pH optimum than the E. coli enzyme.

Cardiolipin synthase from Escherichia coli.

Escherichia coli cardiolipin synthase catalyzes reversible phosphatidyl group transfer from one phosphatidylglycerol molecule to another to form cardiolipin (CL) and glycerol. The enzyme is specified by the cls gene, located at min 28.02 of the E. coli genetic map. Cells with mutations in cls have longer doubling times, tend to lose viability in the stationary phase, are more resistant to 3,4-dihydroxybutyl-1-phosphonate, and have an altered sensitivity to novobiocin. Although cls null mutants appear to lack CL synthase activity, they are still able to form trace quantities of CL. The enzyme appears to be regulated at both the genetic and enzymatic levels. CL synthase's molecular mass is 45-46 kDa, or about 8 kDa less than the polypeptide predicted by the gene sequence, suggesting that posttranslational processing occurs. CL synthase can use various polyols such as mannitol and arabitol to convert CL to the corresponding phosphatidylglycerol analog. When the amino acid sequences of four bacterial CL synthases are compared, three highly conserved regions are apparent. One of these regions contains a conserved pentapeptide sequence, RN(Q)HRK, and another has a conserved HXK sequence. These two sequences may be part of the active site. E. coli CL synthase has been studied by using a mixed micelle assay. The enzyme is inhibited by CL, the product of the reaction, and by phosphatidate. Phosphatidylethanolamine partially offsets inhibition caused by CL but not by phosphatidate. CDP-diacylglycerol does not appear to affect the activity of the purified enzyme but does stimulate the activity associated with crude membrane preparations.

Identity of the Escherichia coli cls and nov genes.

cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene. A comparison of the DNA sequences of both genes further verifies their identity.

The effects of phosphoglycerides on Escherichia coli cardiolipin synthase.

Escherichia coli cardiolipin synthase catalyzes the conversion of two phosphatidylglycerol molecules to cardiolipin and glycerol. This enzyme was amplified in strain BL21(DE3) bearing recombinant plasmid pLR3, which was itself constructed by inserting the cls gene downstream from a T7 RNA promoter. Membranes from BL21(DE3)/pLR3 have over 1200 times more cardiolipin synthase activity than do comparable membranes from wild type cells. The enzyme was purified to homogeneity by extraction with Triton X-114 and chromatography on DEAE-cellulose. The purified enzyme migrated as a single band (46 kDa) on SDS-PAGE. This, along with SDS-PAGE analysis of induced protein, supports the notion that cls is the structural gene for cardiolipin synthase. Cardiolipin synthase activity was determined in a mixed micelle assay in which phosphatidyl[2-3H]glycerol was the substrate. The enzyme is inhibited by the product of the reaction, cardiolipin, and by phosphatidate. However, it is not inhibited by two other anionic phosphoglycerides, phosphatidylinositol and bis-phosphatidate. Phosphatidylethanolamine partially offsets inhibition by cardiolipin but not by phosphatidate. Magnesium chloride has the opposite effect. Cardiolipin inhibition of cardiolipin synthase probably plays an important role in regulating cardiolipin synthesis in E. coli.

The phosphonic acid analog of phosphatidylglycerol phosphate: influence on Escherichia coli growth and physiology.

At 20 microM, rac-3,4-dihydroxybutyl-1-phosphonate (DBP) has only a slight bacteriostatic effect on Escherichia coli. However, cells lose viability when the medium also contains either 20 mM magnesium or calcium ions. Magnesium ions stimulate the incorporation of DBP into (1,2-diacyl)-sn-glycerol-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-pho sphonate, the phosphonate analog of phosphatidylglycerol phosphate. Much higher DBP concentrations are needed to block the growth of a pgsA3 mutant than to block the growth of an isogenic wild-type strain. The DBP-treated pgsA mutant also has a much higher survival rate when stored in the cold than does the DBP-treated wild-type strain. Furthermore, the pgsA3 mutant grows normally in the presence of DBP and magnesium ions. Treatment with DBP and magnesium ions does not appear to disrupt the cell's inner or outer membranes. However, it does block macromolecular and phosphoglyceride synthesis. A combination of 20 microM rac-DBP and 0.5 mM spermidine or 0.125 mM spermine is bacteriostatic. These studies indicate that the PGP analog contributes to DBP's bacteriostatic effect when the growth medium contains low concentrations of magnesium or calcium ions and is responsible for its bactericidal effect when the medium contains high concentrations of these ions.

Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.

Genetic regulation of cardiolipin synthase in Escherichia coli.

Recombinant DNA and genetic techniques were used to construct Escherichia coli strains SOH92 [phi(cls-lacZ+)] and SOH93 [phi(cls-'lacZ)hyb]. beta-Galactosidase (116 kDa) synthesized by strain SOH92 was primarily present in the particulate fraction. Strain SOH92 produced about 20-fold more beta-galactosidase activity than strain SOH93. Expression of clsphilacZ in both SOH92 and SOH93 was influenced by the terminal electron acceptor (increasing in the order oxygen, nitrate, fumarate) when the cells were cultured in minimal medium with glycerol as the sole carbon source. As strains SOH92 and SOH93 progressed from early to late log growth phase under aerobic conditions in LB broth, clsphilacZ expression increased about 2.5-fold. Fusion strains containing a pss-1 allele had an increased cardiolipin (CL) level, but no corresponding increase in clsphilacZ expression was observed. A cls::Tn10dTet null mutation was introduced into SOH92 and SOH93. The strains produced less CL, but no corresponding changes in clsphilacZ expression were observed. A high copy number plasmid bearing the cls gene had no effect on clsphilacZ expression. Taken together, these results indicate that cls is not subject to autogenous regulation.

Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli.

When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH oxidoreductase had an apparent Km of 28 and 35 microM for L-GAP and NADPH, respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6. The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent Km of 280 microM.

Hepatic fat accumulation during liver regeneration.

Fat accumulates in the regenerating liver after partial hepatectomy. The role of intracellular acyltransferases in the synthesis of triglycerides was studied in the liver. Two-thirds hepatectomy was performed in 14 rats. An additional 14 rats had laparotomy only (sham group). Animals were sacrificed at 18 or 24 hr after operation. After 18 hr, microsomal sn-glycerol 3-phosphate acyltransferase specific activity was increased from 708 to 956 pmole of glycerol 3-phosphate incorporated X min-1 X mg-1 protein and the triglyceride content was increased from 2.2 to 12.6 mg/g liver. At 24 hr the microsomal enzyme activity was again increased from 742 to 1203 pmole of glycerol 3-phosphate incorporated X min-1 X mg-1 protein and the triglyceride content rose from 1.9 to 13.9 mg/g liver. A correlation existed between the microsomal enzyme activity and the triglyceride level (r = 0.608, P less than 0.05). The mitochondrial enzyme activity was not increased. At 24 hr peroxisomal dihydroxyacetone acyltransferase activity was elevated from 375 to 523 pmole of dihydroxyacetone phosphate incorporated X min-1 X mg-1 protein but showed no correlation with the triglyceride content; the microsomal activity of the enzyme was not increased. Cytoplasmic NAD+-dependent alpha-glycerol 3-phosphate dehydrogenase decreased by 24 and 32% at 18 and 24 hr. These data indicate that the microsomal sn-glycerol 3-phosphate acyltransferase activity has a major role in promoting triglyceride synthesis during liver regeneration.

Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli.

Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected. This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP. One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction. Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition. The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination. Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP. The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations. All phenotypic effects attributed to strain 6204 appear to be due to a single mutation. Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min. Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp. The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls. Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls. Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective. These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis. To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP. These strains had growth properties that were identical with those of strain 6204. Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride. Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.

Effects of halo analogs of glycerol 3-phosphate and dihydroxyacetone phosphate upon Escherichia coli.

The fluoro and chloro analogs of glycerol 3-phosphate inhibit the growth of Escherichia coli and affect bacterial enzymes involved in lipid synthesis.

Uptake of glycerol 3-phosphate and some of its analogs by the hexose phosphate transport system of Escherichia coli.

The hexose phosphate transport system transported glycerol 3-phosphate and its analogs 3,4-dihydroxybutyl-1-phosphonate, glyceraldehyde 3-phosphate, and 3-hydroxy-4-oxobutyl-1-phosphonate.

Effect of 3,4-dihydroxybutyl-1-phosphonate on phosphoglyceride and lipoteichoic acid synthesis in Bacillus subtilis.

3,4-Dihydroxybutyl-1-phosphonate (CH2OHCHOHCH2CH2PO3HPO3H2), an analogue of glycerol 3-phosphate, preferentially inhibits the rate of synthesis and accumulation of phosphatidylglycerol in Bacillus subtilis W23 and 168. The rate of phosphatidylethanolamine synthesis is only slightly inhibited, whereas that of lysylphosphatidylglycerol is somewhat stimulated. As expected, decreased phosphatidylglycerol synthesis results in the inhibition of the formation of the putative lipoteichoic acid precursor, sn-glycero-1-phospho-beta-gentiobiosyldiacyglycerol and of lipoteichoic acid itself.

Biosynthesis of murein lipoprotein in Escherichia coli: effects of 3,4-dihydroxybutyl-1-phosphonate.

The effects of 3,4-dihydroxybutyl-1-phosphonate, a four-carbon analog of sn-glycerol 3-phosphate, on the biosynthesis of the glyceryl moiety in murein lipoprotein of Escherichia coli were studied. The compound at a concentration of 55 microM strong inhibits in the incorporation of [2-3H]glycerol radioactivity into lipoprotein by virtue of its inhibition of the synthesis of phosphatidylglycerol. On the other hand, the incorporation of prelabeled [2-3H]glycerol radioactivity into lipoprotein was only partially inhbited by 3,4-dihydroxybutyl-1-phosphonate even at a much higher concentration (1 mM). These data were consistent with the postulated pathway for the biosynthesis of the lipid moiety in lipoportein: cysteine-lipoprotein + phosphatidylglycerol leads to glycerylcystein-lipoprotein + phosphatidic acid.

Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism.

3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system. There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected. Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected. Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size. The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase. A Km mutant for the former enzyme was susceptible to the phosphansferase activity. Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action.

Synthesis of phosphonate and ether analogs of rac-phosphatidyl-L-serine.

The chemical synthesis of four phosphonate-containing phosphatidylserine analogs namely, L-serine (+/-)-[2,3-bis(hexadecyloxy) and 2,3-bis(Palmitoyloxy)-propyl] phosphonates, and L-serine (+/-)-[3,4-bis(hexadecyloxy and 3,4-bis(palmitoyloxy)-butyL]phosphonates is descirbed. (+/-)-2,3-Bis(hexadecyloxy) and 2,3-bis(palmitoyloxy)-prophylphosphonic acids and (+/-)-3,4-bis(hexadecyloxy)butylphosponic acid were prepared by reaction of tris(trimethylsily) phosphite on the corresponding haloalkane. Condensation of the above phosphonic acids or (+/-)-3,4-bis(palmitoyloxy)butylphosphonic acid with N-carboxy-L-serine dibenzyl ester in the presence of trichloroacetonitrile or triisopropylbenzenesulfonyl chloride yielded the protected serine intermediates, which on hydrogenolysis gave the desired L-serine analogs. By a similar route, 1,2-dihexadecyl-rac-glycero-3-phosphoric acid was converted to 1,2-dihexadecyl-rac-glycerophospho-L-serine (L-serine (+/-)-2,3-bis(hexadecyloxy)propyl hydrogen phosphate(ester).

Isosteres of natural phosphates. 5. The preparation of phosphotidycholine, phosphotidylethanolamine and phosphotidylglycerol.

Isosteric phosphonate analogues of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol have been prepared in which the normal oxygen atom of the phosphate ester linkage in the glycerol backbone of the molecule has been replaced by a methylene group. These have been prepared by the coupling of the previously reported phosphotidic acid (isosteric analogue of phosphatidic acid) with the appropriate protected hydroxyl compound mediated by trichloroacetonitrile. In all cases, racemic materials have been isolated: for the analogue of phosphatidylglycerol, a mixture of two enantiomeric pairs is isolated.

Isosteres of natural phosphates. 6. The preparation and properties of lysophosphotidic acid.

We herein report the first chemical synthesis of phosphonic acid analogues of lysophosphatidic acid. The racemic isosteric analogues, 4-acyloxy-3-hydroxybutyl-1-phosphonic acids, of lysophosphatidic acid were prepared by both catalytic and hydride reductions of the 4-acyloxy-3-oxobutyl-1-phosphonic acids, a general method for the preparation of the latter having been reported previously. The lysophosphatidic acids have been found to substrates for lysophosphatidic acid acyl transferase, and may be acylated chemically to yield phosphotidic acids. The latter reaction is of use in the preparations of differentially acylated phosphatidic acids.