The Biology and Enzymology of ProteinN-Myristoylation (2001)

N-myristoyltransferase Protein N-myristoylation refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine of eukaryotic and viral proteins.N-Myristoylproteins have diverse functions and intracellular destinations. They include proteins involved in a wide variety of signal transduction cascades. In general, N-myristoylation is an irreversible protein modification that occurs co-translationally following removal of the initiator methionine residue by cellular methionylaminopeptidases (1Wolven A. Okamura H. Rosenblatt Y. Resh M.D. Mol. Biol. Cell. 1997; 8: 1159-1173Crossref PubMed Scopus (146) Google Scholar, 2Towler D.A. Adams S.P. Eubanks S.R. Towery D.S. Jackson-Machelski E. Glaser L. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2708-2712Crossref PubMed Scopus (169) Google Scholar). N-Myristoylation can also occur post-translationally, as in the case of the pro-apoptotic protein BID where proteolytic cleavage by caspase 8 reveals a “hidden” myristoylation motif (3Zha J. Weiler S. Oh K.J. Wei M.C. Korsmeyer S.J. Science. 2000; 290: 1761-1765Crossref PubMed Scopus (471) Google Scholar). N-Myristoylation promotes weak and reversible protein-membrane and protein-protein interactions (4Peitzsch R.M. McLaughlin S. Biochemistry. 1993; 32: 10436-10443Crossref PubMed Scopus (459) Google Scholar, 5Murray D. Ben-Tal N. Honig B. McLaughlin S. Structure. 1997; 5: 985-989Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Typically, myristate acts in concert with other mechanisms to regulate protein targeting and function. Some proteins (e.g. MARCKS, Src) employ “myristoyl-electrostatic switches” where membrane association is promoted by myristate plus electrostatic interactions between positively charged protein side chains and negatively charged membrane phospholipids (6McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (612) Google Scholar, 7Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1066) Google Scholar). In other N-myristoylproteins, ligand binding (Arfs, recoverin (8Goldberg J. Cell. 1998; 95: 237-248Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 9Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (426) Google Scholar)) or proteolytic cleavage (HIV-1 Pr55gag/p17MA (10Hermida-Matsumoto L. Resh M.D. J. Virol. 1999; 73: 1902-1908Crossref PubMed Google Scholar)) produces a conformational change that exposes or sequesters the acyl chain (“myristoyl-conformational switch”). A subset of proteins undergoes post-translational covalent modification with one or more palmitoyl groups after N-myristoylation. Examples of these dually acylated proteins include members of the Src family of tyrosine kinases (Fyn, Lck), α subunits of heterotrimeric G-proteins, endothelial nitric-oxide synthase, and the yeast vacuolar protein Vac8p (11Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (312) Google Scholar). Cys serves as the acceptor site for palmitate.S-Palmitoylation is reversible, providing a mechanism for regulated interactions between these N-myristoylproteins and cellular membranes and/or other proteins (7Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1066) Google Scholar, 12Linder M.E. Tamanoi F. Sigman D.S. The Enzymes: Protein Lipidation. Academic Press, Inc., San Diego, CA2000: 215-240Google Scholar). For example, turnover of the palmitoyl moiety in N-myristoylated Giα subunits is stimulated by activation of the 5-hydroxytryptamine-1a receptor (13Chen C.A. Manning D.R. J. Biol. Chem. 2000; 275: 23516-23522Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Depalmitoylation of Giα has no overt effects on membrane association (14Huang C. Duncan J.A. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 412-417Crossref PubMed Scopus (83) Google Scholar) but may have functional consequences for G-protein de-activation; in vitro studies have shown that S-acylated Giα is resistant to the GTPase-activating protein activity of RGS (regulators of G-protein signaling) proteins (15Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). The “kinetic bilayer trapping” hypothesis (16Shahinian S. Silvius J.R. Biochemistry. 1995; 34: 3813-3822Crossref PubMed Scopus (261) Google Scholar) was proposed to explain the operation of this “dual fatty acylation switch.” According to the hypothesis, proteins with myristate are able to transiently interact with membranes. This allows subsequent palmitoylation by plasma membrane-associated palmitoylS-transferase (17Dunphy J.T. Greentree W.K. Manahan C.L. Linder M.E. J. Biol. Chem. 1996; 271: 7154-7159Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and stable membrane association because of a slower off rate of the dually acylated product. Dual acylation also influences compartmentalization to specialized plasmalemmal microdomains (18Oh P. Schnitzer J.E. Mol. Biol. Cell. 2001; 12: 685-698Crossref PubMed Scopus (346) Google Scholar). In lymphocytes, dual acylation of non-receptor tyrosine kinases (Fyn, Lck) is necessary for their targeting to lipid rafts. Inhibiting S-acylation by Cys to Ser mutation, or 2-bromopalmitate treatment, delocalizes these kinases from rafts and is associated with loss of signaling through the T-cell receptor (19Kabouridis P.S. Magee A.I. Ley S.C. EMBO J. 1997; 16: 4983-4998Crossref PubMed Scopus (315) Google Scholar, 20Webb Y. Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Mutagenesis studies indicate that both N-myristoylation andS-palmitoylation are needed to target endothelial nitric-oxide synthase to caveolae (21Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). N-Myristoylation is catalyzed by myristoyl-CoA:proteinN-myristoyltransferase (Nmt),1 a member of the GNAT superfamily of proteins (22Dyda F. Klein D.C. Hickman A.B. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 81-103Crossref PubMed Scopus (369) Google Scholar). Nineteen Nmts have been identified from 15 species (see supplemental material for alignments plus a phylogenetic analysis). Genetic studies have established the essential requirement for Nmt in a number of these species. TheSaccharomyces cerevisiae genome contains a singleNMT gene. A nmt1 null allele causes recessive lethality (23Duronio R.J. Towler D.A. Heuckeroth R.O. Gordon J.I. Science. 1989; 243: 796-800Crossref PubMed Scopus (156) Google Scholar). Arabidopsis thaliana contains twoNMT genes. Forced expression of Nmt1 cRNA produces stunted growth and reduced survival of transgenic plants (24Qi Q. Rajala R.V. Anderson W. Jiang C. Rozwadowski K. Selvaraj G. Sharma R. Datla R. J. Biol. Chem. 2000; 275: 9673-9683Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A null mutation of Drosophila melanogaster dNMT results in embryonic lethality (25Ntwasa M. Aapies S. Schiffmann D.A. Gay N.J. Exp. Cell Res. 2001; 262: 134-144Crossref PubMed Scopus (41) Google Scholar). Mutant embryos display a range of phenotypes, including morphogenetic abnormalities associated with disordered cell movement and widespread apoptosis. Some of these changes phenocopy changes produced by genetic manipulations affecting D. melanogaster N-myristoylproteins involved in dynamic rearrangements of the actin cytoskeleton, e.g. non-receptor tyrosine kinases (Dsrc42A, Dsrc64B). Finally, a NMT null allele causes lethality in the two principal causes of systemic fungal infections in immunocompromised humans, Candida albicans andCryptococcus neoformans (26Weinberg R.A. McWherter C.A. Freeman S.K. Wood D.C. Gordon J.I. Lee S.C. Mol. Microbiol. 1995; 16: 241-250Crossref PubMed Scopus (116) Google Scholar, 27Lodge J.K. Jackson-Machelski E. Toffaletti D.L. Perfect J.R. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12008-12012Crossref PubMed Scopus (169) Google Scholar). Three mammalian species (human, mouse, Bos taurus) each possess two Nmts (termed type I and type II (28Giang D.K. Cravatt B.F. J. Biol. Chem. 1998; 273: 6595-6598Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar)). Type I Nmts show a high degree of conservation across species, as do the orthologous type II Nmts. Type I and II Nmts are most divergent at their N termini (see alignment in the supplemental material). Glover et al. (29Glover C.J. Hartman K.D. Felsted R.L. J. Biol. Chem. 1997; 272: 28680-28689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) reported that the extended N-terminal domain of type I human Nmt may be involved in targeting the enzyme to the ribosome but is not required for activity in vitro. The divergent N-terminal domains of the two Nmt isoforms may allow differential cellular localization, thereby effecting co-translational ribosome-based or post-translational cytosol-based protein acylation. Nmt represents an attractive therapeutic target given its requirement for the survival of several human pathogens. Different classes of Nmt inhibitors have been identified including analogs of myristate and myristoyl-CoA (30Paige L.A. Zheng G.Q. DeFrees S.A. Cassady J.M. Geahlen R.L. J. Med. Chem. 1989; 32: 1665-1667Crossref PubMed Scopus (42) Google Scholar, 31Paige L.A. Zheng G.Q. DeFrees S.A. Cassady J.M. Geahlen R.L. Biochemistry. 1990; 29: 10566-10573Crossref PubMed Scopus (70) Google Scholar, 32Devadas B. Lu T. Katoh A. Kishore N.S. Wade A.C. Mehta P.P. Rudnick D.A. Bryant M.L. Adams S.P. Li Q. Gokel G.W. Gordon J.I. J. Biol. Chem. 1992; 267: 7224-7239Abstract Full Text PDF PubMed Google Scholar, 33Cordo S.M. Candurra N.A. Damonte E.B. Microbes Infect. 1999; 1: 609-614Crossref PubMed Scopus (41) Google Scholar, 34Harper D.R. Gilbert R.L. Blunt C. McIlhinney R.A. J. Gen. Virol. 1993; 74: 1181-1184Crossref PubMed Scopus (30) Google Scholar, 35Saermark T. Kleinschmidt A. Wulff A.M. Andreassen H. Magee A. Erfle V. AIDS. 1991; 5: 951-958Crossref PubMed Scopus (17) Google Scholar, 36Nadler M.J. Harrison M.L. Ashendel C.L. Cassady J.M. Geahlen R.L. Biochemistry. 1993; 32: 9250-9255Crossref PubMed Scopus (42) Google Scholar), myristoylpeptide derivatives (2Towler D.A. Adams S.P. Eubanks S.R. Towery D.S. Jackson-Machelski E. Glaser L. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2708-2712Crossref PubMed Scopus (169) Google Scholar,37Furuishi K. Matsuoka H. Takama M. Takahashi I. Misumi S. Shoji S. Biochem. Biophys. Res. Commun. 1997; 237: 504-511Crossref PubMed Scopus (17) Google Scholar), and histidine analogs (38Raju R.V. Datla R.S. Warrington R.C. Sharma R.K. Biochemistry. 1998; 37: 14928-14936Crossref PubMed Scopus (15) Google Scholar). All studied Nmts exhibit a preference for myristoyl-CoA. However, they have divergent peptide substrate specificities. Therefore, peptide derivatives offer an opportunity to generate species-selective inhibitors. A large library of peptidomimetics has been developed through depeptidization of an octapeptide representing the N-terminal sequence of a known yeast Nmt substrate, Arf2p (GLYASKLS, i.e. Ref. 39Devadas B. Zupec M.E. Freeman S.K. Brown D.L. Nagarajan S. Sikorski J.A. McWherter C.A. Getman D.P. Gordon J.I. J. Med. Chem. 1995; 38: 1837-1840Crossref PubMed Scopus (56) Google Scholar). Some members of this compound library have fungistatic or fungicidal activity against C. albicans and C. neoformans(40Lodge J.K. Jackson-Machelski E. Devadas B. Zupec M.E. Getman D.P. Kishore N. Freeman S.K. McWherter C.A. Sikorski J.A. Gordon J.I. Microbiology. 1997; 143: 357-366Crossref PubMed Scopus (35) Google Scholar, 41Lodge J.K. Jackson-Machelski E. Higgins M. McWherter C.A. Sikorski J.A. Devadas B. Gordon J.I. J. Biol. Chem. 1998; 273: 12482-12491Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 42Devadas B. Freeman S.K. McWherter C.A. Kishore N.S. Lodge J.K. Jackson-Machelski E. Gordon J.I. Sikorski J.A. J. Med. Chem. 1998; 41: 996-1000Crossref PubMed Scopus (49) Google Scholar). S. cerevisiae Nmt (Nmt1p) is the best studied of the known Nmts. It is a monomer with no known co-factor requirements or post-translational modifications. Nmt1p is highly selective for myristoyl-CoA in vitro and in vivo. In vitrokinetic studies of synthetic peptides derived from the N-terminal sequences of known N-myristoylproteins have produced a set of empiric rules for identifying candidate substrates. The following criteria, based on these empiric rules, were used to search theS. cerevisiae genome data base for known or putative Nmt substrates: (a) Gly absolutely required at the +1 position; (b) charged residues, aromatics and Pro not allowed at +2; (c) all amino acids allowed at +3 and +4; (d) Ser, Thr, Ala, Gly, Cys, or Asn permitted at +5; and (e) all but Pro allowed at +6. Seventy-one ORFs (1% of the total) were identified. Among these proteins, there was a marked preference for Ser at position +5 (37/71) and for Lys at +6 (16/71). Steady state kinetic, isothermal titration calorimetric, and x-ray crystallographic studies (43Rudnick D.A. McWherter C.A. Rocque W.J. Lennon P.J. Getman D.P. Gordon J.I. J. Biol. Chem. 1991; 266: 9732-9739Abstract Full Text PDF PubMed Google Scholar, 44Bhatnagar R.S. Schall O.F. Jackson-Machelski E. Sikorski J.A. Devadas B. Gokel G.W. Gordon J.I. Biochemistry. 1997; 36: 6700-6708Crossref PubMed Scopus (40) Google Scholar, 45Farazi T.A. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 6335-6343Crossref PubMed Scopus (62) Google Scholar) all support the conclusion that Nmt1p follows an Ordered Bi Bi reaction mechanism. The apo-enzyme binds myristoyl-CoA to form a Nmt1p·myristoyl-CoA binary complex that is competent to acquire peptide substrates. Conversion of the enzyme·substrate complex to the enzyme·product complex (chemical transformation) is followed by release of CoA and then myristoylpeptide. Pre-steady state kinetic analyses indicate that the rate-determining step occurs after the chemical transformation,i.e. the rate of chemical transformation is 50–200-fold faster than the overall rate, depending upon the peptide substrate (46Farazi T.A. Manchester J.K. Gordon J.I. Biochemistry. 2000; 39: 15807-15816Crossref PubMed Scopus (20) Google Scholar). Measuring enzymatic rates in the presence of increasing buffer microviscosity (47Farazi T.A. Manchester J.K. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 9177-9186Crossref PubMed Scopus (23) Google Scholar), as well as structural studies of Nmt1p complexed with substrates and substrate analogs (45Farazi T.A. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 6335-6343Crossref PubMed Scopus (62) Google Scholar), suggest that this step corresponds to a conformational change that allows product release (see below). Structural studies of Nmt have provided insights about how it catalyzes N-myristoylation through a direct nucleophilic addition-elimination reaction. The x-ray structure of C. albicans apo-Nmt has been determined to 2.45-Å resolution (48Weston S.A. Camble R. Colls J. Rosenbrock G. Taylor I. Egerton M. Tucker A.D. Tunnicliffe A. Mistry A. Mancia F. de la Fortelle E. Irwin J. Bricogne G. Pauptit R.A. Nat. Struct. Biol. 1998; 5: 213-221Crossref PubMed Scopus (102) Google Scholar), as have the structures of an S. cerevisiaeNmt1p·myristoyl-CoA binary complex (2.2 Å (45Farazi T.A. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 6335-6343Crossref PubMed Scopus (62) Google Scholar)) and two ternary complexes: Nmt1p·S-(2-oxo)pentadecyl-CoA·SC-58272 (2.9 Å (49Bhatnagar R.S. Futterer K. Farazi T.A. Korolev S. Murray C.L. Jackson-Machelski E. Gokel G.W. Gordon J.I. Waksman G. Nat. Struct. Biol. 1998; 5: 1091-1097Crossref PubMed Scopus (106) Google Scholar)) and Nmt1p·S-(2-oxo)pentadecyl-CoA·GLYASKLA (2.5 Å (45Farazi T.A. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 6335-6343Crossref PubMed Scopus (62) Google Scholar)). S-(2-oxo)pentadecyl-CoA is a non-hydrolyzable myristoyl-CoA analog with a methylene group interposed between the reactive thioester carbonyl and sulfur (30Paige L.A. Zheng G.Q. DeFrees S.A. Cassady J.M. Geahlen R.L. J. Med. Chem. 1989; 32: 1665-1667Crossref PubMed Scopus (42) Google Scholar). It is a potent competitive inhibitor of S. cerevisiae Nmt1p (K i = 5 nm) that binds to the apo-enzyme with a similar thermodynamic signature as myristoyl-CoA (44Bhatnagar R.S. Schall O.F. Jackson-Machelski E. Sikorski J.A. Devadas B. Gokel G.W. Gordon J.I. Biochemistry. 1997; 36: 6700-6708Crossref PubMed Scopus (40) Google Scholar). SC-58272 is a potent competitive peptidomimetic inhibitor (K i = 43 nm) derived from the Arf2p-related octapeptide substrate GLYASKLA. SC58272 retains two elements critical for recognition (Ser, Lys) but replaces the N-terminal GLYA with a 2-methylimidazole plus a rigidified carbon linker (p-(2-methylimidazole-N-butyl)phenylacetyl) and the C-terminal LA with cyclohexylethyl (39Devadas B. Zupec M.E. Freeman S.K. Brown D.L. Nagarajan S. Sikorski J.A. McWherter C.A. Getman D.P. Gordon J.I. J. Med. Chem. 1995; 38: 1837-1840Crossref PubMed Scopus (56) Google Scholar). The Nmt1p fold consists of a saddle-shaped β-sheet flanked by α helices. There is pseudo-2-fold symmetry. The N-terminal half forms the myristoyl-CoA binding site. The C-terminal half forms the bulk of the peptide binding site (Fig.1A). Each half has a fold similar to the core structure of GNAT superfamily members (22Dyda F. Klein D.C. Hickman A.B. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 81-103Crossref PubMed Scopus (369) Google Scholar). Residues 1–54 in C. albicans apo-Nmt and 1–33 in the binary and ternary Nmt1p complexes are disordered. Binding of myristoyl-CoA to apo-Nmt1p induces two conformational changes: (a) formation of a 310 helix (A′) from part of the disordered N terminus to complete the myristoyl-CoA binding site (Fig. 1 B) and (b) a change in the loop connecting helix A and strand b to open a “lid” overlying the peptide binding site (see Ab loop in Fig. 1 C). The pantetheine of CoA forms an important part of this peptide binding site (Fig.2C). Bound myristoyl-CoA has a conformation resembling a question mark (Fig.2 A). As described below, this conformation may facilitate product release. The thioester carbonyl of myristoyl-CoA is H-bonded to the backbone amides of Phe-170 and Leu-171 (Fig. 2 A). These two residues, positioned in a bulge in β strand e, form an oxyanion hole that polarizes the carbonyl. (Oxyanion holes are present at analogous positions in other GNATs; see Protein Data Bank accession numbers 1CJW, 1QSN, 1BO4 and Ref. 22Dyda F. Klein D.C. Hickman A.B. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 81-103Crossref PubMed Scopus (369) Google Scholar.) Nmtlp uses the elements that produce bends at C-1 and C-6 of myristate, as well as the floor of its acyl-CoA binding pocket, as key landmarks to measure and properly position myristoyl-CoA. The binding site structure indicates that the two additional methylenes of palmitoyl-CoA could not be accommodated without affecting positioning of its C-1 carbonyl in the oxyanion hole. Kinetic studies of stopped flow tryptophan fluorescence reveal that myristoyl-CoA binding occurs as a (46Farazi T.A. Manchester J.K. Gordon J.I. Biochemistry. 2000; 39: 15807-15816Crossref PubMed Scopus (20) Google Scholar). The is most The is of myristoyl-CoA and most represents the rate of formation of the N-terminal 310 Nmt can be from other GNAT family members by the of its peptide substrates. The reported structural about interactions between a Nmt and its peptide substrates from the ternary structure with GLYASKLA. is positioned Å from the thioester carbonyl of myristoyl-CoA in the resolution a that can be by 2 Å after (see Fig. and plus the of mechanism below). The several the is Å from the of Å from the of and Å from the of (Fig. between the side chain of of the peptide and pantetheine of myristoyl-CoA complete formation of the peptide binding site and at the generate a in the peptide it from myristoyl-CoA and a peptide binding (Fig. 2 is accommodated in a with in results in a in the chemical transformation rate, that this residue is important in positioning the reactive (47Farazi T.A. Manchester J.K. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 9177-9186Crossref PubMed Scopus (23) Google Scholar). is accommodated in an of from the site of Nmt in a large that could (Fig. 2 is in yeast N-myristoylproteins, is H-bonded to the side chain of as well as the backbone amides of and (Fig. 2 also in yeast N-myristoylproteins, is H-bonded to the side chain of its is by the of and and the of (Fig. 2 The structural for in the peptide substrate of orthologous and human Nmts to be The described in S. cerevisiae Nmt1p that interact with are highly However, and form part of a involved in of in are by Ser and Ala, in the two human Nmts. This that human Nmt could and/or more at position of its substrates. Kinetic studies of stopped flow tryptophan fluorescence have shown that binding of peptide substrate to the binary complex is a that is most (46Farazi T.A. Manchester J.K. Gordon J.I. Biochemistry. 2000; 39: 15807-15816Crossref PubMed Scopus (20) Google Scholar). Binding to a change in the conformation of the Ab loop that serves to the “lid” on the peptide binding site interact with the of and 2 In the ternary complex of Nmt1p the Ab loop forms additional with the This that the of of the Ab loop may with the overall of Nmt1p substrates. SC58272 functions as a potent at in because it is able to form a complex with with the Ab These have for species-selective inhibitors (see below). Structural with state and state kinetic analyses of Nmt1p (47Farazi T.A. Manchester J.K. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 9177-9186Crossref PubMed Scopus (23) Google Scholar) have identified several elements in the site of the enzyme that facilitate the reaction. The proposed reaction is by a in the supplemental The oxyanion by the backbone amides of Phe-170 and polarizes the reactive carbonyl of myristoyl-CoA to subsequent nucleophilic by the N-terminal of a peptide The C-terminal of positioned Å from the N-terminal Gly of the acceptor functions as the base that the Gly to a nucleophilic of the C-terminal of Nmt1p results in a in the chemical transformation rate and the step of the enzyme from a step after chemical transformation to the transformation (47Farazi T.A. Manchester J.K. Waksman G. Gordon J.I. Biochemistry. 2001; 40: 9177-9186Crossref PubMed Scopus (23) Google Scholar). In other GNAT a side chain functions as a base to facilitate nucleophilic of a amino group on the thioester carbonyl of Y. N.A. R. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), this is by the backbone carbonyl of in S. cerevisiae 1 C. D. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). A of the the between it and the thioester carbonyl of myristoyl-CoA from to Å (Fig. and interactions between the and position the the reaction and facilitate nucleophilic The oxyanion hole with the by and the of the to of this the is the group of CoA is through between these two groups (see the in supplemental material). The group is by the of the CoA This mechanism is and for the conformation of a CoA facilitate from the site. Steady state with increasing of with the x-ray crystallographic studies of the binary and ternary Nmt complexes suggest that the step in the reaction is an The could of the changes that occur upon myristoyl-CoA of the N-terminal 310 helix and the conformation of the Ab the of the step of Nmt additional kinetic as well as structural (e.g. The may for Nmt inhibitors for protein N-myristoylation in cell and/or pathogens. analogs that the preference of orthologous Nmts for myristoyl-CoA and their divergent peptide substrate may species-selective and may be by that the conformation and of the Ab loop of the A key but question is how Nmt its substrates and this additional protein The of two Nmt isoforms with divergent N termini the that this domain may enzyme activity by recognition or interactions with substrates or the intracellular of Gokel and Linder for and for the of the proposed mechanism of with with