Abstract
The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNA(Gln) and a glutaminyl-tRNA amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln). Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyl-tRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria.
Highlights
Plasmodium apicoplast protein synthesis is essential for parasite survival, yet few of the enzymes involved have been biochemically characterized
The P. falciparum genome contains two single-exon genes that encode putative orthologs of the GatA and GatB subunits of the bacterial glutamyl-tRNA amidotransferase (Glu-AdT) as follows: GatA (PF3D7_0416100, 96 kDa, 826 amino acids) and GatB (PF3D7_0628800, 102 kDa, 882 amino acids) (Table 1). Both P. falciparum proteins possess predicted N-terminal bipartite apicoplast targeting sequences, suggesting that they are the subunits of an amidotransferase that participates in an apicoplast indirect aminoacylation pathway
All bacterial GatCAB enzymes studied to date are able to serve as both a glutamyl-tRNAGln amidotransferase (Glu-AdT) and an aspartyltRNAAsn amidotransferase (Asp-AdT) in vitro (64 – 69)
Summary
Plasmodium apicoplast protein synthesis is essential for parasite survival, yet few of the enzymes involved have been biochemically characterized. Results: Nucleus-encoded apicoplast GatAB glutamyl-tRNA amidotransferase forms Gln-tRNAGln in concert with a nondiscriminating glutamyl-tRNA synthetase. Conclusion: Formation of apicoplast Gln-tRNAGln is via indirect aminoacylation. The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNAGln and a glutaminyl-tRNA amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln. Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyltRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNAGln to Gln-tRNAGln in vitro. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Plasmodium GatA appears to have emerged prior to the bacterial-archaeal phylogenetic divide. GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria
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