Abstract - Shevyreva

Phosphatidylcholine (PC), a typical eukaryotic phospholipid, is an important lipid compound of some bacterial membranes as well. The amount of PC in bacteria ranges from a few percent of total membrane lipids in Xanthomonas campestris to nearly 70% in Acetobacter aceti. Notably, in certain pathogenic or symbiotic bacteria, PC plays a crucial role in facilitating interactions with their eukaryotic hosts.
One of the most common PC biosynthesis pathways in bacteria is a three-step S-adenosylmethionine (SAM)-dependent methylation of phosphatidylethanolamine (PE) to PC mediated by a single or a combination of multiple phospholipid N-methyltransferases (Pmts). Pmts are classified based on their sequence similarities into the Sinorhizobium (S) and Rhodobacter (R) types. These enzymes display different substrate preferences regardless of their type. For instance, the S-type Agrobacterium tumefaciens PmtA (AtPmtA) catalyzes all three methylations of PE to produce PC, while another S-type enzyme, Rhizobium leguminosarum PmtS1, can execute only the initial step of the pathway. Although AtPmtA has been extensively studied biochemically, and recent structural insights have been gained about both an R-type representative from Rubellimicrobium thermophilum (RtPmtA) and AtPmtA, understanding of the catalytic mechanism in bacterial Pmts remains insufficient.
The main goal of this project was the elucidation and comparative analysis of the catalytic mechanisms of S-type and R-type Pmts. For this purpose, we employed a combination of computational and biochemical research.
Based on our findings, we suggest that despite the low amino acid sequence identity, the two Pmt classes exhibit similar reaction mechanisms with tyrosine residues mainly involved in the catalysis of methyl group transfer. However, the number and localization of these key residues vary between the two Pmt types. RtPmtA features two highly conserved tyrosines in the substrate-binding pocket, located on the N-terminal αA-helix. In contrast, in the S-type AtPmtA, only one tyrosine, positioned in the protein core, has an impact on the enzyme´s activity. Furthermore, based on the structural analysis of RtPmtA and AtPmtA, we propose that the specific active site modifications indicate distinct mechanisms for tyrosine activation, thereby illustrating the evolutionary divergence of the two Pmt types.

Irina Shevyreva, Lena Sophie Fritsch, Leander Mika Koch, Anton Effing, Meriyem Aktas and Franz Narberhaus*