Toward understanding the catalytic mechanism for two classes of bacterial phospholipid <i>N</i> ‐methyltransferases

Phosphatidylcholine (PC), the predominant phospholipid in eukaryotic membranes, also plays a crucial role in certain bacterial species, often mediating interactions with eukaryotic hosts. In bacteria, a major pathway for PC biosynthesis involves the three-step methylation of phosphatidylethanolamine, catalyzed by phospholipid N-methyltransferases (Pmts). While the binding site for the methyl donor S-adenosyl-l-methionine is well characterized in Pmt enzymes, the detailed mechanism of methyl group transfer remains poorly understood. In this study, we combined computational and biochemical approaches to identify the amino acid residues critical for the catalytic activity of two distinct Pmt classes: the Rhodobacter (R)-type enzyme from Rubellimicrobium thermophilum (RtPmtA) and the Sinorhizobium (S)-type enzyme from Agrobacterium tumefaciens (AtPmtA). Despite low sequence identity, both enzyme types share similar reaction mechanisms, with tyrosine residues playing key roles in methyl group transfer. In RtPmtA, two highly conserved tyrosines located within the substrate-binding pocket on the N-terminal αA-helix are critical for enzymatic function. In contrast, AtPmtA depends on a single tyrosine buried in the protein core for catalysis. These findings reveal distinct active site architectures and suggest that R-type and S-type enzymes have evolved class-specific structural strategies for tyrosine activation. This divergence highlights the evolutionary flexibility of Pmt enzymes, despite their shared catalytic function.