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 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 </=50% of total cellular Arf1p.
Zhang
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Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. jgordon@pharmdec.wustl.edu
Jackson-Machelski
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Gordon
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D000217:Acyltransferases / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D003545:Cysteine; D005656:Fungal Proteins / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D005656:Fungal Proteins; D010455:Peptides; D006639:Histidine; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D003545:Cysteine