Molecular cloning, genomic organization, and biochemical characterization of myristoyl-CoA : protein N-myristoyltransferase from Arabidopsis thaliana

Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes t he co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions. We have is olated a full-length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the f irst described from a higher plant, This AtNMT1 cDNA clone has an open read ing frame of 434 amino acids and a predicted molecular mass of 48,706 Da, T he primary structure is 50% identical to the mammalian NMTs, Analyses of So uthern blots, genomic clones, and database sequences suggested that the A. thaliana genome contains two copies of NMT gene, which are present on diffe rent chromosomes and have distinct genomic organizations. The recombinant A tMMT1 expressed in Escherichia coli exhibited a high catalytic efficiency f or the peptides derived from putative plant myristoylated proteins AtCDPK6 and Fen kinase, The AtNMT was similar to the mammalian NMTs with respect to a relative specificity for myristoyl CoA amoung the acyl CoA donors and al so inhibition by the bovine brain NMT inhibitor NIP71. The AtNMT1 expressio n profile indicated ubiquity in roots, stem, leaves, flowers, and siliques (approximate to 1.7 kb transcript and approximate to 50 kDa immunoreactive polypeptide) but a greater level in the younger tissue, which are developme ntally very active, NMT activity was also evident in all these tissues. Sub cellular distribution studies indicated that, in leaf extracts, similar to 60% of AtNMT activity was associated with the ribosomal fractions, whereas similar to 30% of the activity was observed in the cytosolic fractions. The NMT is biologically important to plants, as noted from the stunted develop ment when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter. The results presented in this st udy provide the first direct molecular evidence for plant protein N-myristo ylation and a mechanistic basis for understanding the role of this protein modification in plants.