Disease relevance of NMT1

• By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A) [1].
• Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I [2].
• Human myristoyl-CoA synthetase and myristoyl-CoA:protein N-myristoyltransferase (hNmt) have been partially purified from an erythroleukemia cell line [3].
• These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor [4].
• 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 [5].

High impact information on NMT1

• Insertional mutagenesis of the NMT1 locus on yeast chromosome XII caused recessive lethality, indicating that this protein acyltransferase activity is necessary for vegetative cell growth [6].
• Overexpression of NMT activity was achieved by means of the yeast episomal plasmid YEp24 without obvious effects on growth kinetics, cell morphology, or acylprotein metabolic labeling patterns [6].
• We show that Gpa1 is myristoylated by Nmt1, and without this normally stable modification, Gpa1 is unable to inhibit pheromone signaling [7].
• Surprisingly, polymerases were detected 4.3 kb beyond the nmt1 polyadenylation [poly(A)] site and 2.4 kb beyond the nmt2 poly(A) site, which in each case have transcribed through an entire convergent downstream transcription unit [8].
• 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 [9].

Chemical compound and disease context of NMT1

• Use of Escherichia coli strains containing fad mutations plus a triple plasmid expression system to study the import of myristate, its activation by Saccharomyces cerevisiae acyl-CoA synthetase, and its utilization by S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase [10].
• Studies of strains that contain a mutation in FadE which inhibits beta-oxidation of exogenous fatty acids, confirm that Nmt1p retains its specificity for myristoyl-CoA over palmitoyl-CoA in E. coli [10].
• Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids [1].

Biological context of NMT1

• Isogenic, haploid strains containing NMT1, nmt1-72, and nmt1-181 do not manifest any obvious differences in steady state levels of the acyltransferases during growth at permissive temperatures or in the biosynthesis of long chain saturated acyl-CoAs [11].
• When compared with wild type Nmt1p, Cys217 --> Arg produces 3- and 6-fold increases in Ki for SC-58272 at 24 and 37 degrees C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site [12].
• Wild type C. neoformans, C. albicans, and H. sapiens NMTs can fully complement the lethal phenotype of a S. cerevisiae nmt1 null allele at 24 and 37 degrees C when the GAL1-10 promoter controlling their expression is induced by galactose [13].
• To identify Nmt1p substrates that may affect maintenance of proliferative potential during stationary phase, we searched the yeast genome for known and putative N-myristoylproteins [14].
• 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 [15].

Anatomical context of NMT1

• Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides [16].
• N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells [17].
• Concerning the enzyme itself, there are evidences that NMT is also present in the endoplasmic reticulum and that its substrate specificity is different from that of the cytosolic enzyme(s) [18].

Associations of NMT1 with chemical compounds

• 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 [19].
• Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates [12].
• 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 [9].
• When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids [9].
• Our recent studies of the 455-amino acid Saccharomyces cerevisiae acyltransferase (Nmt1p) suggested that its mechanism of catalysis is ordered Bi Bi with myristoyl-CoA binding occurring prior to binding of peptide and release of CoA occurring prior to release of the myristoyl-peptide [20].

Other interactions of NMT1

• 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 [19].
• Although substitution of nmt1-451D for NMT1 results in deficiencies in protein N-myristoylation, these deficiencies are modest and limited by compensatory responses that include augmented expression of nmt1-451D and precocious induction of FAA4 in log phase [14].
• The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to </=50% of total cellular Arf1p [12].
• FAS1 expression is Ino2p-dependent in NMT1 cells at 24-33 degreesC [21].
• Comparative analysis of the beta transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1 [22].

Analytical, diagnostic and therapeutic context of NMT1

• To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene [12].
• 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 [15].
• Isothermal titration calorimetric studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase. Determinants of binding energy and catalytic discrimination among acyl-CoA and peptide ligands [23].
• The molecular structure of the ternary complex of Saccharomyces cerevisiae Nmt1p with a bound non-hydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, and a competitive peptidomimetic inhibitor, SC-58272, was solved to 2.9 A resolution by X-ray crystallography [24].
• Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania [25].

References