Emily Jackson-Machelski

Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs.

N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins. It is a target for anti-fungal and anti-viral therapy. We have determined the structure, to 2.9 Å resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzymes substrate specificities and regulate the ordered binding and release of substrates and products. A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzymes C-terminal backbone carboxylate.

N-myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA: protein N-myristoyltransferase.

Myristoyl-CoA: protein N-myristoyltransferase (Nmt) catalyses the covalent attachment of myristate to the N-terminal glycine of a small subset of cellular proteins produced during vegetative growth of Candida albicans. nmt447D is a mutant NMT allele encoding an enzyme with a Gly447-->ASP substitution and reduced affinity for myristoyl-CoA. Among isogenic NMT/NMT, NMT/ delta nmt and nmt delta/nmt447D strains, only nmt delta/nmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 degrees C. When switched from YPD/myristate to YPD alone, 60% of the organisms die with 4 h. Antibodies raised against the C-terminal eight residues of Saccharomyces cerevisiae Arf1p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains. N-Myristoylation of C. albicans ADP-ribosylation factor (Arf) produced a change in its electrophoretic mobility during SDS-PAGE: the myristoylated species migrated more rapidly than the nonmyristoylated species. In an NMT/nmt delta strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmt delta/nmt447D cells were incubated at 24 degrees C in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, > or = 50% of total cellular Arf was nonmyristoylated. This finding suggests that > or = 50% reduction in Arf N-myristoylation is a biochemical marker of a growth-arrested cell. A similar conclusion was made after assaying isogenic S. cerevisiae strains containing various combinations of NMT1, nmt1-451D, ARF1, arf1 delta, ARF2 and arf2 delta alleles and grown at 24-37 degrees C on YPD of YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevisiae Aft. SC-59383 has an IC50 of 1.45 +/- 0.08 microM for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransferase. It had an EC50 of 51 +/- 17 and 67 +/- 6 microM, 24 and 48 h after a single administration of the drug to cultures of C. albicans. The Arf gel mobility shift assay indicated that a single dose of 200 microM produced a < 50% reduction in Arf N-myristoylation after 4 h, which is consistent with the fungistatic, but not fungicidal, activity. The effect on Nmt was specific: an enantiomer, SC-59840, had no inhibitory effect on purified C. albicans Nmt (IC50 > 1,000 microM), and 200 microM of the compound produced no detectable reduction in Arf N-myristoylation in vivo. SC-58272, which is related to SC-59383, was a more potent inhibitor in vitro (IC50 0.056 +/- 0.01 microM), but had no growth inhibitory activity and did not produce any detectable reduction in Arf N-myristoylation. These findings highlight the utility of the Arf protein gel mobility shift assay for demonstrating the mechanism-based antifungal activity of SC-59383, a selective inhibitor of C. albicans Nmt.

Genetic and Biochemical Studies Establish That the Fungicidal Effect of a Fully Depeptidized Inhibitor of Cryptococcus neoformans Myristoyl-CoA:ProteinN-Myristoyltransferase (Nmt) Is Nmt-dependent

Cryptococcus neoformans is a fungal pathogen that causes chronic meningitis in 10% of patients with AIDS. Genetic and biochemical studies were conducted to determine whether myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a target for development of a new class of fungicidal drugs. A single copy of a conditional lethal C. neoformans NMT allele was introduced into the fungal genome by homologous recombination. The allele (nmt487D) produces temperature-sensitive myristic acid auxotrophy. This phenotype is due, in part, to under-myristoylation of a cellular ADP ribosylation factor (Arf) and can be rescued by forced expression of human Nmt. Two isogenic strains with identical growth kinetics at 35 °C were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 containednmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 °C, survival of HMC1 depends upon its human Nmt. ALYASKLS-NH2, an inhibitor derived from an Arf, was fully depeptidized:p-[(2-methyl-1-imidazol-1-yl)butyl]phenyl-acetyl was used to represent the GLYA tetrapeptide, whereas SKLS was replaced with a chiral tyrosinol scaffold. Kinetic studies revealedK i  (app) values of 1.8 ± 1 and 9 ± 2.4 μm for purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was 2-fold lower for CPA8 compared with HMC1. A single dose of 100 μm produced a 5-fold greater inhibition of protein synthesis in CPA8 versus HMC1. The strain specificity of these responses indicates that the fungicidal effect was Nmt-dependent. These two strains may be useful for screening chemical libraries for Nmt-based fungicidal compounds with relatively little activity against the human enzyme.

Biochemical Studies of Saccharomyces cerevisiae Myristoyl-coenzyme A:Protein N-Myristoyltransferase Mutants

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins. Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates. To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene. A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1 alleles encoding enzymes with reduced activity. nmt1 alleles were identified that produced temperature-sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn102, Ala202, Cys217, Ser328, Val395, Asn404, Leu420, and Asn426. Ala202 → Thr, Cys217 → Arg, Ser328 → Pro, Asn404 → Tyr, and Asn426 → Ile produced the most severe ts phenotype. Their effects on the functional properties of the enzymes myristoyl-CoA and peptide binding sites were defined by purifying each mutant from Escherichia coli and conducting in vitro kinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p). None of the substitutions affect the enzymes acyl chain length selectivity. When compared with wild type Nmt1p, Cys217 → Arg produces 3- and 6-fold increases in Ki for SC-58272 at 24 and 37°C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1ps peptide binding site. Asn426 → Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold). Ala202 → Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the Ki for S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the Ki for SC-58272 at 24 and 37°C. An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions. The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to ≤50% of total cellular Arf1p.