Structural insights into phosphatidylethanolamine formation in bacterial membrane biogenesis

Area of Science:

• Structural Biology
• Biochemistry
• Microbiology

Background:

• Phosphatidylethanolamine (PE) is a crucial cellular membrane component synthesized by phosphatidylserine decarboxylase (PSD) in bacteria.
• The activation and substrate binding mechanisms of PSD, particularly its auto-cleavage and pyruvoyl cofactor utilization, remain structurally uncharacterized.
• Understanding PSD's structure is key to elucidating PE biosynthesis and bacterial membrane homeostasis.

Purpose of the Study:

• To determine the high-resolution X-ray crystal structures of Escherichia coli PSD.
• To elucidate the structural basis for PSD auto-cleavage, substrate binding, and catalytic mechanism.
• To investigate the enzyme's interaction with lipid bilayers and its membrane association topology.

Main Methods:

• X-ray crystallography was employed to obtain structures of apo-PSD and a PE-bound complex.
• The PE-bound complex mimics the Schiff base intermediate formed during decarboxylation.
• Site-directed mutagenesis was used to identify key residues involved in auto-cleavage.

Main Results:

• High-resolution structures reveal PE-bound PSD with the phospholipid conjugated to the pyruvoyl group, mimicking the catalytic intermediate.
• Extensive hydrophobic interactions between PSD and PE's fatty acyl chains explain the enzyme's broad substrate specificity.
• PSD adopts a monotopic membrane association via its N-terminal amphipathic helices, positioning it within the lipid bilayer.
• Mutagenesis identified D90, D142, H144, and S254 as critical for PSD auto-cleavage and proenzyme maturation.

Conclusions:

• The structures provide unprecedented insights into the mechanism of PE synthesis by PSD.
• PSD's unique N-terminal domain facilitates monotopic membrane association, crucial for its function in the lipid bilayer.
• The identified catalytic residues (D90/D142-H144-S254) are essential for enzyme activation via auto-cleavage.