No abstract

The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic constructs that display potent inhibition of bacterial growth, with activities approaching that of nisin itself. Most notable was the activity observed against clinically relevant bacterial strains including MRSA and VRE. Experiments with membrane models indicate that these constructs operate via a lipid II-mediated mode of action without causing pore formation. A lipid II-dependent mechanism of action is further supported by antagonization assays wherein the addition of lipid II was found to effectively block the antibacterial activity of the nisin-derived lipopeptides.

No abstract

Protein arginine N-methyltransferases (PRMTs) catalyze methyl-group transfer from S-adenosyl-L-methionine onto arginine residues in proteins. In this study, modifications were introduced at the guanidine moiety of a peptidyl arginine residue to investigate how changes to the PRMT substrate can modulate enzyme activity. We found that peptides bearing Nη-hydroxy or Nη-amino substituted arginine showed higher apparent kcat values than for the monomethylated substrate when using PRMT1, whereas this catalytic preference was not observed for PRMT4 and PRMT6. Methylation by compromised PRMT1 variants E153Q and D51N further supports the finding that the N-hydroxy substitution facilitates methyl transfer by tuning the reactivity of the guanidine moiety. In contrast, Nη-nitro and Nη-canavanine substituted substrates inhibit PRMT activity. These findings demonstrate that methylation of these PRMT substrates is dependent on the nature of the modification at the guanidine moiety.

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved KM values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.