Mll5 is a SET domain protein that appears to lack histone methyltransferase activity. (A) Recombinant Mll5 does not reveal in vitro histone methyltransferase activity toward core histones, isolated from calf thymus, or to nucleosomes isolated from Saccharomyces cerevisiae. Recombinant Mll5 lacking its SET domain and HsSUV39H1 were used as negative and positive controls, respectively. (B) Alignment of the catalytic region within the SET domain of various mouse proteins and Drosophila and S cerevisiae MmMll5 homologs CG9007, SET3, and SET4, respectively. The 2 adjacent amino acids asparagine (N) and histidine (H), which are part of one of the most conserved sequence motifs in the SET domain (R-F/Y-I-N-H-X-C-X-P-N) and are invariant in all SET domains with established lysine methyltransferase activity, are altered to arginines in Mll5. The same substitutions are found in Drosophila CG9007 and S cerevisiae SET3 and SET4. Interestingly, whereas no information on the enzymatic activities of CG9007 and SET4 is available, specific efforts to detect histone methyltransferase activity for SET3 proved unsuccessful.⁴⁸  Sequence alignment was performed using the Clustal-X program. Colors highlight conservation of different residues among the SET domain proteins. (C) Schematic diagram highlighting amino acids required for catalytic activity, as determined by mutational analyses of the MmSuv39h1 and NcDim5 HMTs. Mutation of the conserved asparagine (N) or histidine (H) amino acids into related (also basic!) residues glutamine (Q) or lysine (K) was sufficient to destroy histone methyltransferase activity of Dim5 and SUV39H1.^49,50  Crystal structure analyses suggest that the conserved N-H residues are critical for binding the methyl-donor S-adenosyl-L-methionine (AdoMet).¹¹  In MmMll5 and the fly and yeast orthologs, these critical residues are replaced by arginines. Taken together, these observations suggest that the SET domain of Mll5 lacks histone methyltransferase activity and might be involved in alternative activities, such as, for example, protein-protein interactions, a proven additional function of the Mll1 SET domain.⁵¹