Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N- myristoylation and cellular lipid metabolism

Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N- myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.     

Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyltransferase deletion mutants and human/yeast Nmt chimeras in Escherichia coli and S. cerevisiae.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, 455-residue, monomeric enzyme. Amino- and carboxyl-terminal deletion mutants of Nmt1p were genetically engineered to determine the minimal domain necessary to maintain catalytic activity. Enzyme activity was assessed by (i) sequentially inducing Nmt1p or its mutant derivatives and one of two eukaryotic substrates for the wild type enzyme (S. cerevisiae Gpa1p and rat Go alpha) in Escherichia coli, a bacterium with no endogenous myristoyltransferase activity, and monitoring Nmt-dependent incorporation of exogenous [3H]myristate into the G protein alpha subunits or (ii) an in vitro enzyme assay using lysates prepared from bacteria producing wild type or mutant Nmts. The data indicate that the minimal catalytic domain of Nmt1p is located between Ile59-->Phe96 and Gly451-->Leu455. Analyses of the ability of mutant nmtps to rescue the lethal phenotype of an nmt1 null allele in a haploid strain of yeast grown on rich media, with or without blockade of cellular fatty acid synthetase, suggest that the amino-terminal 59 residues of Nmt1p may play an important noncatalytic role, functioning as a targeting signal so this cytosolic enzyme can access cellular myristoyl-CoA pools generated from activation of exogenous C14:0 by acyl-CoA synthetase(s). Moreover, there appear to be differences in the location or accessibility of myristoyl-CoA pools derived from fatty acid synthetase and acyl-CoA synthetases. The E. coli co-expression system was used to map structural elements that determine differences in the peptide substrate specificities of Nmt1p and the orthologous human Nmt. Rat Go alpha is a substrate for both enzymes, whereas human Gz alpha is a substrate only for human NMT. Studies of a series of chimeric enzymes composed of elements from the amino- or carboxyl-terminal portions of human and yeast Nmts indicate that (i) recognition/utilization of Gz alpha involves elements distributed from the amino-terminal half through the region defined by Leu352-->Lys410 of the 416 residue human enzyme and (ii) formation of a fully functional peptide binding site and a fully functional myristoyl-CoA binding site in either of these enzymes requires contributions from both their amino-terminal and carboxyl-terminal halves.     

Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N- myristoylation

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.     

Analysis of the compartmentalization of myristoyl-CoA:protein N-myristoyltransferase in Saccharomyces cerevisiae.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational, covalent attachment of a rare fatty acid, myristic acid (C14:0), to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction as well as several viral proteins necessary for assembly-replication. NMT has become a target for both anti-viral and anti-fungal therapy. Analysis of purified Saccharomyces cerevisiae NMT plus yeast strains with conditional lethal nmt1 mutations have provided insights about how this process is regulated in vivo. We have now defined the location of NMT in two strains of S. cerevisiae to better understand the functional and spatial relationships between this enzyme and cellular systems that generate its acyl-CoA and peptide ligands. Western blot studies using an affinity purified antibody raised in rabbits against purified S. cerevisiae NMT indicate that the acyltransferase represents 0.06% of total cellular proteins in an exponentially growing haploid strain with a wild type NMT1 allele. Another strain containing a single, integrated copy of a GAL1/NMT1 fusion gene and a nmt1 null allele had 12-fold higher levels of NMT when grown on galactose-containing media. This increase in NMT production had no detectable effects on growth or cellular morphology. Cell fractionation studies, confocal fluorescence immunocytochemical analysis, and immunogold electron microscopic surveys of fixed, gelatin-embedded cryosections of both strains revealed that NMT is a cytosolic protein that is not associated with cellular membranes (including the endoplasmic reticulum and plasma membrane), the nucleus, mitochondria, Golgi apparatus, or vacuoles. This finding is discussed in light of what is known about the location and activities of enzymes involved in de novo fatty acid biosynthesis and in amino-terminal processing of nascent proteins.     

N-myristoylation regulates the SnRK1 pathway in Arabidopsis

Cotranslational and posttranslational modifications are increasingly recognized as important in the regulation of numerous essential cellular functions. N-myristoylation is a lipid modification ensuring the proper function and intracellular trafficking of proteins involved in many signaling pathways. Arabidopsis thaliana, like human, has two tightly regulated N-myristoyltransferase (NMT) genes, NMT1 and NMT2. Characterization of knockout mutants showed that NMT1 was strictly required for plant viability, whereas NMT2 accelerated flowering. NMT1 impairment induced extremely severe defects in the shoot apical meristem during embryonic development, causing growth arrest after germination. A transgenic plant line with an inducible NMT1 gene demonstrated that NMT1 expression had further effects at later stages. NMT2 did not compensate for NMT1 in the nmt1-1 mutant, but NMT2 overexpression resulted in shoot and root meristem abnormalities. Various data from complementation experiments in the nmt1-1 background, using either yeast or human NMTs, demonstrated a functional link between the developmental arrest of nmt1-1 mutants and the myristoylation state of an extremely small set of protein targets. We show here that protein N-myristoylation is systematically associated with shoot meristem development and that SnRK1 (for SNF1-related kinase) is one of its essential primary targets.     

The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.     

11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase

The covalent attachment of myristic acid to the NH2-terminal glycine residue of proteins is catalyzed by the enzyme myristoyl CoA:protein N-myristoyltransferase (NMT). Using synthetic octapeptide substrates we have identified and characterized an NMT activity in wheat germ lysates used for cell-free translation of exogenous mRNAs. C-12 and C-14 fatty acids are efficiently transferred to the peptides by this plant NMT, but C-10 and C-16 fatty acids are not. Glycine is required as the NH2-terminal residue: peptides with an NH2-terminal alanine were not substrates. Peptides with proline, aspartic acid, or tyrosine residues adjacent to the NH2-terminal glycine were also not myristoylated. Serine in the fifth position reduced the peptide's Km up to 4000-fold. We have chemically synthesized a sulfur analogue of myristate, 11-(ethylthio)undecanoic acid. Its CoA ester is as good a substrate as myristoyl-CoA for both wheat germ and yeast NMT. Peptides linked to 11-(ethylthio)undecanoic acid are less hydrophobic than the corresponding myristoylpeptides. 11-(Ethylthio)-undecanoic acid may, therefore, help define the role of myristic acid in targeting of acyl proteins within cells.     

Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase

Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT). NMT is absolutely specific for peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated. Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT. Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities. The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme.     

A hamster temperature-sensitive alanyl-tRNA synthetase mutant causes degradation of cell-cycle related proteins and apoptosis

We have isolated a temperature-sensitive alanyl-tRNA synthetase mutant from hamster BHK21 cells, designated as ts ET12. It has a single nucleotide mutation, converting the 321st amino acid residue, 321Gly, to Arg. The mutation was localized between two RNA-binding domains of alanyl-tRNA synthetase. Thus far, we have isolated two temperature-sensitive aminoacyl-tRNA synthetase mutants from the BHK21 cell line: ts BN250 and ts BN269. They are defective in histidyl- and lysyl-tRNA synthetase respectively. Both mutants rapidly undergo apoptosis at the nonpermissive temperature, 39.5 degrees C. ts ET12 cells, however, did not undergo apoptosis until 48 h after a temperature-shift to 39.5 degrees C, while mutated alanyl-tRNA synthetase of ts ET12 cells was lost within 4 h. Loss of the mutated alanyl-tRNA synthetase was inhibited by a ubiquitin-dependent proteasome inhibitor, MG132, and by a protein-synthesis inhibitor, cycloheximide. Cell-cycle related proteins were also lost in ts ET12 cells at 39.5 degrees C, as shown in ts BN250. In contrast, the mutated aminoacyl-tRNA synthetases of ts BN250 and ts BN269 were stable at 39.5 degrees C. However, the defects of these mutants released EMAPII, an inducer of apoptosis at 39.5 degrees C. No release of EMAPII occurred in ts ET12 cells at 39.5 degrees C, consistent with the delay of apoptosis in these cells.     

Arginine Auxotrophic Phenotype of Mutations in pyrA of Salmonella typhimurium: Role of N-Acetylornithine in the Maturation of Mutant Carbamylphosphate Synthetase

Mutations in pyrA that abolish catalytic activity of carbamylphosphate synthetase cause auxotrophy for both arginine and a pyrimidine. Eight pyrA mutants auxotrophic only for arginine (AUX) were isolated by the mutagenized phage technique; three of these required arginine only at low temperature (20 degrees C). Explanations of the AUX phenotype based on bradytrophy were eliminated by the discovery that blocking the utilization of carbamylphosphate for pyrimidine biosynthesis by insertion of an additional mutation in pyrB (encoding aspartic transcarbamylase) did not reduce the requirement for arginine. In contrast, mutational blocks in the arginine biosynthetic pathway before N-acetylornithine (argB, argC, argG, or argH) did suppress the mutation in pyrA. This suggests that exogenous arginine permits growth of the AUX mutants by inhibiting the first step in the arginine pathway, thereby preventing accumulation of an intermediate that antagonizes mutant pyrA function. A mutation in argA (N-acetylornithinase) failed to suppress AUX, indicating that N-acetylornithine was the inhibitory intermediate. This intermediate had no effect on the catalytic or regulatory properties of carbamylphosphate synthetase from mutant cells grown under permissive conditions (37 degrees C). However, the regulatory properties of carbamylphosphate synthetase synthesized under restrictive conditions (20 degrees C) were demonstrably defective (insensitive to activation by ornithine); the enzyme synthesized under permissive conditions was activated by ornithine. A strain carrying an additional mutation (argC), which prevents the accumulation of N-acetylornithine, produced an ornithine-activatable enzyme at both growth temperatures. These results suggest that N-acetylornithine antagonizes the proper preconditioning or maturation of the mutant carbamylphosphate synthetase.     

Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 microM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16-66 microM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.     

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

Influence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast Rhodotorula aurantiaca A19, isolated from Antarctic ice, that the inability to grow at temperatures close to 20 degrees C is associated with profound alterations in cell morphology and integrity. High performance liquid chromatography analysis of the intracellular acyl-CoA esters revealed an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated close to the nonpermissive temperature. Its concentration (500 microm) was found to be 28-fold higher than in cells cultivated at 0 degrees C. If one considers its ability to disrupt membrane bilayers and to inhibit many cellular enzymes and functions, intracellular myristoyl-CoA accumulation in the psychrophile R. aurantiaca represents one of the principal causes of growth arrest at moderate temperatures. Intracellular acyl-CoA concentrations are believed to be regulated by thioesterase activity. Thus in an attempt to explore the mechanism by which temperature disrupts myristoyl-CoA metabolism, we isolated and characterized a long chain acyl-CoA thioesterase. The monomeric 80-kDa thioesterase from the psychrophilic yeast shows a very strong specificity for myristoyl-CoA. The affinity for substrate and the catalytic efficiency of the thioesterase are optimal below 5 degrees C (temperatures habitually experienced by the strain) and dramatically decrease with increasing temperature. The loss of affinity for substrate is related to the intracellular increase of myristoyl-CoA concentration. Our observations reveal one of the probable mechanisms by which temperature fixes the limit of growth for this psychrophilic yeast.     

A new,robust, and nonradioactive approach for exploring N-myristoylation

Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.     

Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.     

Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes.     

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.     

A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli.

A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40 degrees C, the strain was fully viable at this temperature as long as guanosine, uridine, histidine, tryptophan, and nicotinamide mononucleotide were all added to the growth medium. Viability of the strain was dependent upon mutations in genes of the nucleoside salvage pathways that improved the utilization of exogenous nucleosides. The properties of the strain are those expected of a PRPP-less strain and suggest that PRPP synthetase is dispensable for E. coli.