Alanyl‐phosphatidylglycerol synthase: mechanism of substrate recognition during tRNA‐dependent lipid modification in Pseudomonas aeruginosa

Abstract

Summary Bacterial lipid homeostasis plays an important role for the adaptation to changing environments and under conditions of antimicrobial treatment. The tRNA-dependent aminoacylation of the phospholipid phosphatidylglycerol catalyzed by aminoacyl-phosphatidylglycerol synthases was shown to render various organisms less susceptible to antibacterial agents. Therefore, this type of enzyme might provide a new target to potentiate the efficacy of existing antimicrobials. This study makes use of the Pseudomonas aeruginosa alanyl-phosphatidylglycerol synthase to identify the minimal core domain of this transmembrane protein which is capable of alanyl-phosphatidylglycerol biosynthesis. Using this catalytic fragment we established a reliable activity assay which was used to study the enzymatic mechanism by analyzing an overall of 33 mutant proteins in vitro. Substrate recognition was analyzed by using aminoacylated microhelices as analogs of the natural tRNA substrate. The enzyme even tolerated mutated versions of this minimal substrate which indicates that neither the intact tRNA, nor the individual sequence of the acceptor stem is a determinant for substrate recognition. Furthermore, the analysis of derivatives of phosphatidylglycerol indicated that the polar headgroup of the phospholipid is specifically recognized by the enzyme, whereas modification of an individual fatty acid or even the deletion of a single fatty acid did not abolish A-PG synthesis.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine

“…These experiments might indicate that the tRNA moiety in combination with the amino acid moiety of Ala-tRNA Ala is relevant for A-PGS substrate recognition. These findings are supported further by earlier experiments (using artificial tRNA microhelices), which revealed the five terminal base pairings as important elements of A-PGS substrate recognition (15).…”

“…2 D and G). Functional relevance for all these polar residues has been confirmed by mutagenesis as summarized in Table S2 (15). Combined interaction of Tyr732/Tyr705 and Asp765/Asp739 with the substrate α-amino group suggests an important role in the recognition of the substrate aminoacyl linkage.…”

“…This domain contains all elements for the specific recognition of the ∼26 kDa water-soluble aminoacyl-tRNA (Ala-tRNA Ala or Lys-tRNA Lys ) and for binding of the hydrophobic PG substrate (15). The N-terminal transmembrane domain anchors aa-PGS…”

“…The aa-PGS enzymes consist of a separable, water-soluble C-terminal domain showing full enzymatic activity (4,15). This domain contains all elements for the specific recognition of the ∼26 kDa water-soluble aminoacyl-tRNA (Ala-tRNA Ala or Lys-tRNA Lys ) and for binding of the hydrophobic PG substrate (15).…”

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…An in-depth study showed that the Ala moiety and the fifth base pair of the tRNA helix constitute the major elements for efficient recognition of Ala-tRNA Ala . 38 Several studies have tried to determine the phenotypical changes correlated with synthesis of Lys-PG compared with Ala-PG in S. aureus and P. aeruginosa . 24,30 These investigations showed that substitution of Lys-PG (which confers a net charge of +1) with Ala-PG (which bears a neutral net charge) did not affect bacterial susceptibility to CAMPs such as nisin and gallidermin, or to the CAMP-like antibiotic daptomycin.…”