Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase

The sn-glycerol-3-phosphate acyltransferase from Escherichia coli, an integral membrane protein whose activity is dependent on phospholipids, was purified to near homogeneity (Green, P. R., Merrill, A. H., Jr., and Bell, R. M., (1981) J. Biol. Chem. 256, 11151-11159). Determination of a partial NH2-terminal sequence and the COOH terminus permitted alignment of the polypeptide on the sequenced sn-glycerol-3-phosphate acyltransferase structural gene (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). Processing of the sn-glycerol-3-phosphate acyltransferase is apparently limited to the removal of the NH2-terminal formylmethionine. Thirteen of 27 possible cyanogen bromide peptides predicted from the DNA sequence were purified, characterized, and assigned to their location in the primary structure. Three peptides located at positions throughout the sequence were partially sequenced by automated Edman degradation. The partial sequence analysis of the homogeneous sn-glycerol-3-phosphate acyltransferase is fully in accord with the primary structure inferred from the DNA sequence.     

A gene (plsD) from Clostridium butyricum that functionally substitutes for the sn-glycerol-3-phosphate acyltransferase gene (plsB) of Escherichia coli.

The sn-glycerol-3-phosphate acyltransferase (plsB) of Escherichia coli is a key regulatory enzyme that catalyzes the first committed step in phospholipid biosynthesis. We report the initial characterization of a novel gene (termed plsD) from Clostridium butyricum, cloned based on its ability to complement the sn-glycerol-3-phosphate auxotrophic phenotype of a plsB mutant strain of E. coli. Unlike the 83-kDa PlsB acyltransferase from E. coli, the predicted plsD open reading frame encoded a protein of 26.5 kDa. Two regions of strong homology to other lipid acyltransferases, including PlsB and PlsC analogs from mammals, plants, yeast, and bacteria, were identified. PlsD was most closely related to the 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) gene family but did not complement the growth of plsC(Ts) mutants. An in vivo metabolic labeling experiment using a plsB plsX plsC(Ts) strain of E. coli confirmed that the plsD expression restored the ability of the cells to synthesize 1-acyl-glycerol-3-phosphate. However, glycerol-3-phosphate acyltransferase activity was not detected in vitro in assays using either acyl-acyl carrier protein or acyl coenzyme A as the substrate.     

sn-Glycerol-3-phosphate auxotrophy of plsB strains of Escherichia coli: evidence that a second mutation, plsX, is required.

sn-Glycerol-3-phosphate auxotrophs defective in phospholipid synthesis contain a Km-defective sn-glycerol-3-phosphate acyltransferase. Detailed genetic analysis revealed that two mutations were required for the auxotrophic phenotype. One mutation, in the previously described plsB locus (sn-glycerol-3-phosphate acyltransferase structural gene), mapped near min 92 on the Escherichia coli linkage map. Isolation of Tn10 insertions cotransducible with the auxotrophy in phage P1 crosses revealed that a second mutation was required with plsB26 to confer the sn-glycerol-3-phosphate auxotrophic phenotype. This second locus, plsX, mapped between pyrC and purB near min 24 on the E. coli linkage map. Tn10 insertions near plsX allowed detailed mapping of the genetic loci in this region. A clockwise gene order putA pyrC flbA flaL flaT plsX fabD ptsG thiK purB was inferred from results of two- and three-factor crosses. Strains harboring the four possible configurations of the mutant and wild-type plsB and plsX loci were constructed. Isogenic plsB+ plsX+, plsB+ plsX50, and plsB26 plsX+ strains grew equally well on glucose minimal medium without sn-glycerol-3-phosphate. In addition, plsX or plsX+ had no apparent effect on sn-glycerol-3-phosphate acyltransferase activity measured in membrane preparations. The molecular basis for the plsX requirement for conferral of sn-glycerol-3-phosphate auxotrophy in these strains remains to be established.     

Isolation of Escherichia coli mutants with elevated levels of membrane enzymes. A trans-acting mutation controlling diglyceride kinase

We have developed a rapid autoradiographic colony assay for detecting mutants with elevated levels of certain biosynthetic enzymes. Four Escherichia coli strains in which the specific activity of the membrane enzyme diglyceride kinase is increased 5-10-fold have been obtained with this approach. The mutant kinase has the same thermal denaturation profile and subcellular localization as the wild type. Five other membrane enzymes involved in phospholipid bilayer assembly are unaffected. In one of these strains (GK-1) the mutation (dgkR-1) responsible for the elevated kinase has been mapped at a new site near minute 92, while the previously identified structural gene (dgk) lies near minute 90. When the structural gene for the kinase (dgk) is cloned on a multi-copy vector-like ColE1, the kinase can be overproduced 5-10-fold on the basis of gene dosage (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) J. Biol. Chem. 255, 9413-9420). Introduction of such hybrid plasmids into a mutant harboring dgkR-1 leads to a multiplicative (rather than additive) effect, resulting in specific activities of diglyceride kinase that are 35-75-fold higher than normal. These results show that dgkR-1 is a trans-acting mutation and suggest the existence of novel regulatory proteins (or metabolites) that direct the expression of certain membrane enzymes.     

Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase. Simultaneous loss of dihydroxyacetone phosphate acyltransferase indicates a common gene

Fourteen independent mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase activity were isolated using a colony autoradiographic screening technique. All 14 mutants were similarly defective in dihydroxyacetone phosphate acyltransferase activity. The mutations were recessive and fell into a single complementation group. Tetrad analysis gave results consistent with mutations in a single nuclear gene affecting both activities. sn-Glycerol-3-phosphate acyltransferase activity from different mutant strains exhibited different substrate dependencies and differing responses to temperature, detergent, and pH. In each case, the response of the dihydroxyacetone phosphate acyltransferase activity was similar to that of the sn-glycerol-3-phosphate acyltransferase. These results are consistent with the mutations occurring in the structural gene. The data also establish that the predominant dihydroxyacetone phosphate acyltransferase activity in yeast is a second activity of the sn-glycerol-3-phosphate acyltransferase.     

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.     

Characterization of active and latent forms of the membrane-associated sn-glycerol-3-phosphate acyltransferase of Escherichia coli

The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution. Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution. Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism. This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column. The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100. Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase. The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme. The physiological significance of these findings is discussed.     

Studies of rat liver microsomal diglyceride acyltransferase and cholinephosphotransferase using microsomal-bound substrate: effects of high fructose intake.

Radiolabeled phosphatidate and diglyceride were prepared bound to rat liver microsomes. These compounds were used as substrates in studies of diglyceride acyltransferase, cholinephosphotransferase, and CTP:phosphatidic acid cytidylyltransferase. Optimum incubation conditions for these reactions in microsomes from normal male rats are described. High fructose diets were fed to rats for 11 days; this resulted in an increased rate of neutral lipid formation from sn-glycerol-3-phosphate by liver microsomal preparations. This was attributed, in part, to a previously reported increase in liver phosphatidate phosphatase activity. The significance of this increase is supported by the finding of a fall in microsomal phosphatidate content and a doubling in microsomal diglyceride. In addition, diglyceride acyltransferase measured with microsomal-bound diglyceride was increased twofold with no equivalent change in cholinephosphotransferase activity. Such a change should result in preferential triglyceride formation from the increased microsomal diglyceride pool. CTP:phosphatidic acid cytidylytransferase activity was depressed by the high fructose diet. These combined alterations would lead to an accelerated hepatic triglyceride formation, a result found in vivo during high fructose feeding. The high fructose diet decreased slightly the total microsomal phospholipid content and markedly depressed phosphatidylethanolamine levels.     

Adenylate kinase of Escherichia coli: evidence for a functional interaction in phospholipid synthesis

Previous genetic and biochemical experiments have suggested that the adenylate kinase of Escherichia coli may be directly involved in phospholipid synthesis through formation of a complex with sn-glycerol-3-phosphate acyltransferase, the membrane-bound enzyme that catalyzes the first step in phospholipid synthesis. In this paper we report direct experiments to test this hypothesis. A mutation within the adenylate kinase structural gene is described that results in a temperature-sensitive phospholipid synthesis (assayed in vivo) and a temperature-sensitive acyltransferase. The adenylate kinase activity of this strain is only minimally altered either in vitro or [as assayed by adenosine 5'-triphosphate (ATP) levels] in vivo. This result demonstrates that the inhibition of phospholipid synthesis is not the result of reduced ATP levels. We report the purification of E. coli adenylate kinase to homogeneity; and find that the addition of homogeneous wild-type adenylate kinase to membranes containing a mutationally altered temperature-sensitive acyltransferase results in thermal stabilization of the acyltransferase activity. Ovalbumin has no such protective effect. Purified E. coli inner membranes contain several proteins that are precipitated by addition of anti adenylate kinase antibody to detergent-solubilized membranes.     

Biosynthesis of inositol in yeast. Primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the INO1 locus

A biochemical, molecular, and genetic analysis of the Saccharomyces cerevisiae INO1 gene and its product, L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been carried out. The sequence of the entire INO1 gene and surrounding regions has been determined. Computer analysis of the DNA sequence revealed four potential peptides. The largest open reading frame of 553 amino acids predicted a peptide with a molecular weight of 62,842. The amino acid composition and amino terminus of purified L-myo-inositol-1-phosphate synthase were chemically determined and compared to the amino acid composition and amino terminus of the protein predicted from the DNA sequence of the large open reading frame. This analysis established that the large open reading frame encodes L-myo-inositol-1-phosphate synthase. The largest of several small open reading frames adjacent to INO1 predicted a protein of 133 amino acids with a molecular weight of 15,182 and features which suggested that the encoded protein may be membrane-associated. A gene disruption was constructed at INO1 by eliminating a portion of the coding sequence and replacing it with another sequence. Strains carrying the gene disruption failed to express any protein cross-reactive to antibody directed against L-myo-inositol-1-phosphate synthase. Although auxotrophic for inositol, strains carrying the gene disruption were completely viable when supplemented with inositol. In a similar fashion, a gene disruption was constructed in the chromosomal locus of the 133-amino acid open reading frame. This mutation did not affect viability but did cause inositol to be excreted from the cell.     

UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme.

The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.     

Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase

Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known. Database searching revealed that the archaeal enzyme shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase IV. The glycerol dehydrogenase, with coordinates that are available today, is closely related to the archaeal enzyme. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the Methanothermobacter thermautotrophicus sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. Based on the model structure, we determined the following: (1) the enzyme requires a Zn(2+) ion for its activity; (2) the enzyme selectively uses the pro-R hydrogen of the NAD(P)H; (3) the putative active site and the reaction mechanism were predicted; and (4) the archaeal enzyme does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase.     

Localization and characterization of the sn-glycerol-3-phosphate acyltransferase in Rhodopseudomonas sphaeroides

The membrane localization and properties of the Rhodopseudomonas sphaeroides sn-glycerol-3-phosphate acyltransferase have been examined utilizing enzymatically prepared acyl-acyl carrier protein (acyl-ACP) substrates as acyl donors for sn-glycerol-3-phosphate acylation. Studies conducted with membranes prepared from chemotrophically and phototrophically grown cells show that sn-glycerol-3-phosphate acyltransferase activity is predominantly (greater than 80%) associated with the cell's cytoplasmic membrane. Enzyme activity associated with the intracytoplasmic membranes present in phototrophically grown R. sphaeroides was within the range attributable to cytoplasmic membrane contamination of this membrane fraction. Enzyme activity was optimal at 40 degrees C and pH 7.0 to 7.5, and required the presence of magnesium. No enzyme activity was observed with any of the long-chain acyl-CoA substrates examined. Vaccenoyl-ACP was the preferred acyl-ACP substrate and vaccenoyl-ACP and palmitoyl-ACP were independently utilized to produce lysophosphatidic and phosphatidic acids. With either vaccenoyl-ACP or palmitoyl-ACP as sole acyl donor substrate, the lysophosphatidic acid formed was primarily 1-acylglycerol-3-phosphate and the Km(app) for sn-glycerol-3-phosphate utilization was 96 microM. The implications of these results to the mode and regulation of phospholipid synthesis in R. sphaeroides are discussed.     

A missense mutation accounts for the defect in the glycerol-3-phosphate acyltransferase expressed in the plsB26 mutant

The sn-glycerol-3-phosphate acyltransferase (plsB) catalyzes the first step in membrane phospholipid formation. A conditional Escherichia coli mutant (plsB26) has a single missense mutation (G1045A) predicting the expression of an acyltransferase with an Ala349Thr substitution. The PlsB26 protein had a significantly reduced glycerol-3-phosphate acyltransferase specific activity coupled with an elevated Km for glycerol-3-phosphate.     

Nucleotide sequence of a cDNA for branched chain acyltransferase with analysis of the deduced protein structure

Nucleotide sequence was determined for a 1.6-kilobase human cDNA putative for the branched chain acyltransferase protein of the branched chain alpha-ketoacid dehydrogenase complex. Translation of the sequence reveals an open reading frame encoding a 315-amino acid protein of molecular weight 35,759 followed by 560 bases of 3'-untranslated sequence. Three repeats of the polyadenylation signal hexamer ATTAAA are present prior to the polyadenylate tail. Within the open reading frame is a 10-amino acid fragment which matches exactly the amino acid sequence around the lipoate-lysine residue in bovine kidney branched chain acyltransferase, thus confirming the identity of the cDNA. Analysis of the deduced protein structure for the human branched chain acyltransferase revealed an organization into domains similar to that reported for the acyltransferase proteins of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. This similarity in organization suggests that a more detailed analysis of the proteins will be required to explain the individual substrate and multienzyme complex specificity shown by these acyltransferases.     

The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

GlpD and PlsB participate in persister cell formation in Escherichia coli

Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.     

Comparison of 1-acylglycerophosphate and glycerophosphate acyltransferases from Euglena microsomes

Both glycerophosphate and monoacylglycerophosphate acyltransferases from Euglena microsomes were inhibited by N-ethylmaleimide, but their responses to heat inactivation and sn-glyceraldehyde-3-phosphate differed. Glycerophosphate acyltransferase had a higher V with palmitoyl-CoA compared to oleoyl-CoA; the reverse was true for monoacylglycerophosphate acyltransferase. Km's (microM) for the glycerophosphate acyltransferase were: palmitoyl-CoA, 21; oleoyl-CoA, 14; and sn-glycerol-3-phosphate, 2900. Km's (microM) for monoacylglycerophosphate acyltransferase were: palmitoyl-CoA, 7; oleoyl-CoA, 4; and 1-palmitoyl-sn-glycerol-3-phosphate, 48.