Authors

Discussion

Gene expression is carefully controlled in response to environmental stimuli in Leishmania. Post-translational modifications (PTMs) have important roles in regulating functions of proteins that carefully control gene expression. Arginine methylation is one such PTM, catalysed by protein arginine methyltransferases (PRMTs). We biochemically characterised PRMT homologs in Leishmania braziliensis: PRMT1, 3, 5, 6 and 7. We recombinantly expressed and purified each of these enzymes and assayed against substrates rich in arginine residues to identify intrinsic methyltransferase activity. LbrPRMT5 and 7 demonstrate strongest activity on an RBP16^102-119 peptide and LbrPRMT7 has broader substrate activity. K_m data measured confirm LbrPRMT7 has highest affinity for the RBP16-derived peptide. LbrPRMT6 demonstrated no activity at all against the substrates tested. Individually, LbrPRMT1 and 3 were inactive against all the peptide substrates tested. Gel filtration data clearly show LbrPRMT1:3 form a hetero-tetrameric complex in solution. This complex was confirmed and characterised via cryo-EM and incubating peptide substrates with LbrPRMT1 and 3 together demonstrates strong methyltransferase activity. Methyltransferase assays with catalytically dead double E loop mutants of LbrPRMT1 and 3 demonstrate LbrPRMT1 is the active component of the complex. Finally, we assess activities of the LbrPRMTs at different temperatures. We show that temperature affects each LbrPRMT in a substrate-specific manner. Combined, our data indicate that LbrPRMT1:3 has a very similar paradigm to TbPRMT1^ENZ:1^PRO, however the retention of a complete double E loop in LbrPRMT3 raises questions about the activity of the LbrPRMT1:3 complex. Therefore, LbrPRMT3 retaining the second glutamate of the double E loop may catalyse an alternative target or serve an unknown function. Moreover, our temperature assay data provides insight into the activities of LbrPRMTs that suggests biologically-relevant, as yet uncharacterised layers of regulation.