Phosphate acyltransferase PlsX is a peripheral membrane protein catalyzing the first chemical step in the phospholipid biosynthesis of many bacteria. Its catalytic function has been demonstrated in a previous study, but its catalytic mechanism has not been scrutinized. Recently, its biological function has been linked to its localization to the newly found membrane microdomains called regions of increased fluidity (RIFs) in Bacillus subtilis. However, little is known about the mechanism of its specific association with the microdomains and how the PlsX-RIFs association affects cell growth. Besides, the dynamics of RIFs is also unclear.

Here, we use a new method to characterize both forward and reverse reactions of the enzyme and show that it is much more efficient than previous...[ Read more ]

Phosphate acyltransferase PlsX is a peripheral membrane protein catalyzing the first chemical step in the phospholipid biosynthesis of many bacteria. Its catalytic function has been demonstrated in a previous study, but its catalytic mechanism has not been scrutinized. Recently, its biological function has been linked to its localization to the newly found membrane microdomains called regions of increased fluidity (RIFs) in Bacillus subtilis. However, little is known about the mechanism of its specific association with the microdomains and how the PlsX-RIFs association affects cell growth. Besides, the dynamics of RIFs is also unclear.

Here, we use a new method to characterize both forward and reverse reactions of the enzyme and show that it is much more efficient than previously recognized and find that the reverse reaction is much faster than the forward reaction. Moreover, the catalytic mechanism of PlsX has also been investigated by docking and mutagenesis studies, indicating that it belongs to a mechanistically unique family of acyl transferases. Additionally, the kinetics and mechanism of glycerol-3-phosphate acyltransferase PlsY from E. coli are also investigated, which exhibits a Ping-Pong mechanism and severe substrate inhibition.

We have also solved a new crystal structure of the protein from B. subtilis and identify a nine-residue amphipathic α-peptide at the exposed end of the four-helix bundle stem in its dimeric structure. We show that this amphipathic α-peptide is responsible for the subcellular localization to RIFs, based on the observation of protein delocalization to the cytosol or other parts of cell membrane when the amphipathicity is disrupted. Further mutational studies show that the short amphipathic α-peptide recognizes RIFs by limiting the hydrophobic interaction in a narrow range close to a minimum level, suggesting that controlling the hydrophobic interaction with the acyl groups of membrane lipids is likely a critical factor in protein recruitment to the fluid membrane microdomains. Furthermore, we determine the putative effect of the RIFs localization on the growth rate of the bacteria and confirm that the PlsX-RIFs interaction is crucial to bacterial growth. Besides, we also identify that RIFs appear during the early-log phase and disappear at the end of mid-log phase, which is closely related to the cell membrane fluidity change.