Topology and Active Site of PlsY: THE BACTERIAL ACYLPHOSPHATE:GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE

• Published in: The Journal of biological chemistry 2007-04, Vol.282 (15), p.11339-11346
• Main Authors: Lu, Ying-Jie, Zhang, Fan, Grimes, Kimberly D, Lee, Richard E, Rock, Charles O
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Bacteria; Binding Sites; Cell Membrane - metabolism; Conserved Sequence; Enzyme Inhibitors - pharmacology; Glycerol-3-Phosphate O-Acyltransferase - antagonists & inhibitors; Glycerol-3-Phosphate O-Acyltransferase - genetics; Glycerol-3-Phosphate O-Acyltransferase - metabolism; Kinetics; Molecular Sequence Data; Mutation - genetics; Streptococcus pneumoniae; Streptococcus pneumoniae - enzymology; Streptococcus pneumoniae - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M700374200

The most widely distributed biosynthetic pathway to initiate phosphatidic acid formation in bacterial membrane phospholipid biosynthesis involves the conversion of acyl-acyl carrier protein to acylphosphate by PlsX and the transfer of the acyl group from acylphosphate to glycerol 3-phosphate by an integral membrane protein, PlsY. The membrane topology of Streptococcus pneumoniae PlsY was determined using the substituted cysteine accessibility method. PlsY has five membrane-spanning segments with the amino terminus and two short loops located on the external face of the membrane. Each of the three larger cytoplasmic domains contains a highly conserved sequence motif. Site-directed mutagenesis revealed that each conserved domain was critical for PlsY catalysis. Motif 1 had an essential serine and arginine residue. Motif 2 had the characteristics of a phosphate-binding loop. Mutations of the conserved glycines in motif 2 to alanines resulted in a Km defect for glycerol 3-phosphate binding leading to the conclusion that this motif corresponded to the glycerol 3-phosphate binding site. Motif 3 contained a conserved histidine and asparagine that were important for activity and a glutamate that was critical to the structural integrity of PlsY. PlsY was noncompetitively inhibited by palmitoyl-CoA. These data define the membrane architecture and the critical active site residues in the PlsY family of bacterial acyltransferases.