Alanyl-Phosphatidylglycerol Synthase from Pseudomonas aeruginosa: Physiological relevance and mechanism of tRNA-dependent catalysis

Abstract

Aminoacyl-phosphatidylglycerol synthases catalyze the tRNA-dependent modification of phosphatidylglycerol (PG) with Ala, Lys and Arg, respectively. However, the exact biological roles and catalytic mechanisms of these enzymes were poorly understood. In this study, the Ala-PG formation in P. aeruginosa PAO1 was investigated. Analysis of the membrane composition of the bacterium grown under acidic conditions revealed 6 % of Ala-PG of the total amount of phospholipids. In agreement, the corresponding PA0920 gene encoding the Ala-PG synthase (A-PGS) was found upregulated under acidic conditions. Formation of Ala-PG was found responsible for the resistance of P. aeruginosa to cefsulodin, Cr3+, sodium lactate, and protamine sulphate. In a second part of this study a P. aeruginosa A-PGS catalytic fragment was produced and employed for the elucidation of the enzymatic mechanism in vitro. Based on a site-directed mutagenesis study with 15 A-PGS mutants a direct transesterification mechanism for A-PGS catalysis was proposed. Determinants of the substrates were elucidated. The polar head group of PG is specifically recognized, whereas the fatty acid residues of PG are not important determinants. The tRNA substrate recognition does not include posttranscriptional base modifications. Besides this, only the acceptor stem of the tRNA is recognized. Based on A-PGS activity assays with tRNA microhelices it was speculated that 5 of the terminal base pairings and especially the C5-G68 base pair are required to direct the alanyl-moiety of Ala-tRNAAla into the active site of the enzyme. Electron microscopic analysis revealed a homogeneous distribution of P. aeruginosa A-PGS in the cytoplasmic membrane. Finally, the localization of the C-terminus of the membrane protein which is relevant for A-PGS activity was determined. Obtained results provided first significant insights into the enzymatic mechanism of A-PG formation which provide a basis for development of A-PGS inhibitory molecules.