Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyltransferase deletion mutants and human/yeast Nmt chimeras in Escherichia coli and S. cerevisiae.

摘要: Abstract Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, 455-residue, monomeric enzyme. Amino- and carboxyl-terminal deletion mutants of Nmt1p were genetically engineered to determine the minimal domain necessary maintain catalytic activity. Enzyme activity was assessed by (i) sequentially inducing or its mutant derivatives one two eukaryotic substrates for wild type enzyme (S. Gpa1p rat Go alpha) in Escherichia coli, a bacterium with no endogenous myristoyltransferase activity, monitoring Nmt-dependent incorporation exogenous [3H]myristate into G protein alpha subunits (ii) vitro assay using lysates prepared from bacteria producing Nmts. The data indicate that located between Ile59-->Phe96 Gly451-->Leu455. Analyses ability nmtps rescue lethal phenotype nmt1 null allele haploid strain yeast grown on rich media, without blockade cellular fatty acid synthetase, suggest amino-terminal 59 residues may play important noncatalytic role, functioning as targeting signal so this cytosolic can access myristoyl-CoA pools generated activation C14:0 acyl-CoA synthetase(s). Moreover, there appear be differences location accessibility derived synthetase synthetases. E. coli co-expression system used map structural elements peptide substrate specificities orthologous human Nmt. Rat both enzymes, whereas Gz only NMT. Studies series chimeric enzymes composed amino- portions Nmts recognition/utilization involves distributed half through region defined Leu352-->Lys410 416 residue formation fully functional binding site either these requires contributions their halves.