Search

Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. 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. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds 16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition site of the enzyme. Moreover, 16−18 have an affinity comparable to that of 2 even though they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal activity.

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 degreesC were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 contained nmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 degreesC, 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 revealed Ki (app) values of 1.8 +/- 1 and 9 +/- 2.4 microM 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 microM 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.

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 enzyme's 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 enzyme's 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 degrees C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's 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 degrees 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

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a “parental” peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.