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.
Cryptococcus neoformans is a major cause of systemic fungal infection in immunocompromised patients. Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyzes the transfer of myristate (C14:0) from myristoyl-CoA to the N-terminal glycine of a subset of cellular proteins produced during vegetative growth of C. neoformans. A Glyw --Asp mutation was introduced into C. neoformans NMT by targeted gene replacement. The resulting strains are temperaturesensitive myristic acid auxotrophs. They are killed at 37C when placed in medium lacking myristate and, in an immunosuppressed animal model ofcryptococcal meningitis, are completely eliminated from the subarachnoid space within 12 days of initial infection. C. neoformans and human Nmts exhibit differences in their peptide substrate specificities. These differences can be exploited to develop a new class of fungicidal drugs.Cryptococcus neoformans var. neoformans is an opportunistic pathogen which has emerged as a serious cause of systemic fungal infection in immunocompromised humans (1-3). Persistent C. neoformans infections are common in AIDS patients after completion of primary therapy with amphotericin B or fluconazole for meningitis. This leads to relapse rates of 50-60%o and a shortened lifespan unless patients receive lifelong suppressive therapy, generally with fluconazole (4, 5). Both amphotericin B and fluconazole target ergosterol, the principal sterol in the organism's plasma membrane. Amphotericin B, a fungicidal macrolide, also binds to cholesterol in animal cell membranes, resulting in renal and other toxicities. Chronic suppressive therapy with the fungistatic drug fluconazole in severely immunocompromised hosts will most likely result in emergence of resistant strains (6). The search for alternative therapeutic targets in this organism would be helped by the ability to use targeted gene disruption to establish whether a given gene product is essential for viability. Protocols for high-efficiency transformation of C. neoformans have been reported (7-9). Gene replacement has remained elusive and may be strain-or locus-specific (9, 10).Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyzes the cotranslational transfer of myristate (C14:0) from myristoyl-CoA to the amino-terminal glycine of a subset of eukaryotic cellular and viral proteins (11). Saccharomyces cerevisiae and human Nmts have an ordered bi-bi reaction mechanism: the apoenzymes bind to myristoyl-CoA, forming a high-affinity binary complex. Formation of this complex allows generation of a functional peptide binding site and subsequent generation of a myristoyl-CoA/Nmt/peptide ternary complex. After catalysis, CoA and then the myristoylpeptide product are released (12-14).NMTgenes have been isolated from S. cerevisiae, Candida albicans, Histoplasma capsulatum, C. neoformans, and humans (15-18). All are present in a single copy per haploid genome. Several S. cerevisiae N-myristoylproteins are essential for viability and depend upon a covalently bound myristoyl group for expression ...
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 A resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzyme's 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 enzyme's C-terminal backbone carboxylate.
Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activit relationships. Expression of the yeast NMT1 gene in Escherchia cofi, 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. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted inE. coliby simultaneous expression ofthe yeastNMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H~palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3HJ10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such Cotranslational (1) covalent attachment of myristic acid (C14:0) to the NH2-terminal glycine residue of a variety of cellular and viral proteins is, in many instances, required for full expression of their biological activity (reviewed in refs. 2 and 3). Current approaches to understanding the contribution of N-myristoylation to protein structure and function have involved site-directed mutagenesis of the NH2-terminal glycine to prevent acylation or the incorporation of heteroatomcontaining analogs of myristic acid with reduced hydrophobicity into N-myristoylated proteins in vivo (4). For example, abolishing myristoylation of the tyrosine kinase p6Ov-src by deletion of its Gly-1 residue or by Gly-1 -+ Ala substitution revealed (5, 6) that the C14:0 fatty acid is required for the protein's stable association with the plasma membrane (probably through interaction with a high-affinity myristoyl-src receptor; refs. 7 and 8) and its ability to transform cells. Analogous mutagenesis of the Gly-1 residues of the Moloney murine leukemia virus Pr65g (9), the Mason-Pfizer monkey virus Pr7859 (10), and the Pr55n of human immunodeficiency virus I (11) blocks viral replication. X-ray crystallographic studies (12) and site-directed mutagenesis (13) of the N-myristoylated poliovirus capsid protein VP4 have also indicated that myristic acid is involved in protein-protein interactions and in vir...
Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that catalyzes the transfer of myristic acid (C14:0) from myristoylCoA to the N-terminus of cellular proteins with a variety of functions. Nmts from an assortment of species display remarkable in vivo specificity for this rare acyl chain. To better understand the mechanisms underlying this specificity, we have used isothermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in chain length and/or conformation, analogs with alterations in the thioester bond, and analogs with or without a 3'-phosphate in their CoA moiety. MyristoylCoA binds to Nmt1p with a Kd of 15 nM and a large exothermic deltaH (-25 kcal/mol). CoA derivatives of C12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but with much smaller deltaH and a correspondingly less negative TdeltaS than myristoylCoA. Replacing the thioester carbonyl group with a methylene or removing the 3'-phosphate of CoA is each sufficient to prevent the low enthalpy binding observed with myristoylCoA. The carbonyl and the 3'-phosphate have distinct and important roles in chain length recognition over the range C12-C16. Acyltransferase activity parallels binding enthalpy. The naturally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCoA are used as alternative Nmt substrates in retinal photoreceptor cells, even though they do not exhibit in vitro kinetic or thermodynamic properties that are superior to those of myristoylCoA. The binding of an acylCoA is the first step in the enzyme's ordered reaction mechanism. Our findings suggest that within cells, limitation of Nmt substrate usage occurs through control of acylCoA availability. This indicates that full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substrates but also consideration of the synthesis and/or presentation of its lipid substrates.
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