The elongation factors of Saccharomyces cerevisiae are extensively methylated, containing a total of ten methyllysine residues. Elongation factor methyltransferases (Efm1, Efm2, Efm3, and Efm4) catalyze at least four of these modifications. Here we report the identification of a new type of protein lysine methyltransferase, Efm5 (Ygr001c), which was initially classified as N6-adenine DNA methyltransferase-like. Efm5 is required for trimethylation of Lys-79 on EF1A. We directly show the loss of this modification in efm5 Delta strains by both mass spectrometry and amino acid analysis. Close homologs of Efm5 are found in vertebrates, invertebrates, and plants, although some fungal species apparently lack this enzyme. This suggests possible unique functions of this modification in S. cerevisiae and higher eukaryotes. The misannotation of Efm5 was due to the presence of a DPPF sequence in post-Motif II, typically associated with DNA methylation. Further analysis of this motif and others like it demonstrates a potential consensus sequence for N-methyltransferases. (C) 2014 Elsevier Inc. All rights reserved.