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 the co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions. We have isolated a full-length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the first described from a higher plant, This AtNMT1 cDNA clone has an open reading frame of 434 amino acids and a predicted molecular mass of 48,706 Da, The primary structure is 50% identical to the mammalian NMTs, Analyses of Southern blots, genomic clones, and database sequences suggested that the A. thaliana genome contains two copies of NMT gene, which are present on different chromosomes and have distinct genomic organizations. The recombinant AtMMT1 expressed in Escherichia coli exhibited a high catalytic efficiency for 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 also inhibition by the bovine brain NMT inhibitor NIP71. The AtNMT1 expression 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 developmentally very active, NMT activity was also evident in all these tissues. Subcellular 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 development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter. The results presented in this study provide the first direct molecular evidence for plant protein N-myristoylation and a mechanistic basis for understanding the role of this protein modification in plants.