The ribosome is one of life’s most important molecular machines. Recent discoveries have positioned it as an important player in translational regulation. One such mechanism through which it may alter its rate of function is through post translational modifications. The human protein methyltransferase SMYD5 has recently been found to catalyse mono-, di-, and tri-methylation of ribosomal protein RPL40 on the K22 residue in humans. Natively, there is no methylation of the K22 residue in Saccharomyces cerevisiae. Here we investigated the role of human SMYD5-mediated methylation of the RPL40 homolog in yeast. With liquid chromatography-tandem mass spectrometry, we showed that S. cerevisiae RPL40 can be methylated by recombinantly expressed SMYD5. However, we found the stoichiometry of RPL40 methylation on its yeast substrate to be significantly lower than in humans. We hypothesised that this may be due to steric clashing of the methyltransferase with its substrate as the human and yeast RPL40 proteins are not sequence identical. We produced computationally resolved structures of a SMYD5 and yeast RPL40 interaction to assess differences in residues at their interface. Position 120 of RPL40, which is asparagine in humans and glutamine in yeast was found to have proximity to SMYD5 at position 349. SMYD5 was mutated at residue I349 to a valine, a smaller residue, to reduce steric clashing and potentially increase SMYD5 activity on RPL40 and increase the stoichiometry of methylation. We intend to further investigate phenotypic changes in S. cerevisiae under various stressors, with a particular interest in antibiotics, how the global rate of translation changes, and how translational fidelity is impacted by SMYD5-mediated RPL40 methylation.