Structural insights into the committed step of bacterial phospholipid biosynthesis

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• PlsY is a crucial enzyme in bacterial phospholipid biosynthesis, catalyzing the acylation of glycerol 3-phosphate (G3P).
• Unlike other acyltransferases, PlsY lacks common motifs, has no eukaryotic homologs, and utilizes acyl-phosphate as an unusual acyl donor.
• Inhibitors of PlsY have shown potential as antimicrobial agents, highlighting its importance as a drug target.

Purpose of the Study:

• To determine the high-resolution crystal structure of PlsY.
• To elucidate the atomic details of PlsY's active site and its mechanism of action.
• To provide structural insights for the development of novel antimicrobial inhibitors targeting PlsY.

Main Methods:

• X-ray crystallography was employed to determine the crystal structure of PlsY at 1.48 Å resolution.
• Substrate- and product-bound structures were obtained to visualize the enzyme's active site.
• Site-directed mutagenesis and a high-throughput enzymatic assay were utilized to investigate the catalytic mechanism and screen for inhibitors.

Main Results:

• The crystal structure revealed PlsY adopts a seven-transmembrane helix fold.
• Analysis of substrate- and product-bound structures identified a relatively inflexible active site.
• Structure and mutagenesis data suggest a 'substrate-assisted catalysis' mechanism, distinct from other acyltransferases, which does not require a protein catalytic base.
• A high-throughput enzymatic assay was developed for inhibitor screening.

Conclusions:

• The determined structure of PlsY provides atomic-level insights into its unique fold and active site.
• The proposed 'substrate-assisted catalysis' mechanism offers a novel understanding of acyltransferase function.
• The structural data and developed assay are valuable resources for the discovery of new antimicrobial agents targeting PlsY.