Dataset Information

Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites.

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.

SUBMITTER: Mailu BM

PROVIDER: S-EPMC4705961 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Publications

Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites.

Mailu Boniface M BM Li Ling L Arthur Jen J Nelson Todd M TM Ramasamy Gowthaman G Fritz-Wolf Karin K Becker Katja K Gardner Malcolm J MJ

The Journal of biological chemistry 20150828 49

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 b ...[more]

PMID: 26318454

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Project description:Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast organelle of malaria parasites. The Fd/FNR system provides reducing power to various iron-sulfur cluster (FeS)-dependent proteins in the apicoplast and is believed to help to maintain redox balance in the organelle. While the Fd/FNR system has been pursued as a target for antimalarial drug discovery, Fd, FNR, and the FeS proteins presumably reliant on their reducing power play an unknown role in parasite survival and apicoplast maintenance. To address these questions, we generated genetic deletions of these proteins in a parasite line containing an apicoplast bypass system. Through these deletions, we discovered that Fd, FNR, and certain FeS proteins are essential for parasite survival but found that none are required for apicoplast maintenance. Additionally, we addressed the question of how Fd and its downstream FeS proteins obtain FeS cofactors by deleting the FeS transfer proteins SufA and NfuApi. While individual deletions of these proteins revealed their dispensability, double deletion resulted in synthetic lethality, demonstrating a redundant role in providing FeS clusters to Fd and other essential FeS proteins. Our data support a model in which the reducing power from the Fd/FNR system to certain downstream FeS proteins is essential for the survival of blood-stage malaria parasites but not for organelle maintenance, while other FeS proteins are dispensable for this stage of parasite development. IMPORTANCE Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form one of the few known redox systems in the apicoplast of malaria parasites and provide reducing power to iron-sulfur (FeS) cluster proteins within the organelle. While the Fd/FNR system has been explored as a drug target, the essentiality and roles of this system and the identity of its downstream FeS proteins have not been determined. To answer these questions, we generated deletions of these proteins in an apicoplast metabolic bypass line (PfMev) and determined the minimal set of proteins required for parasite survival. Moving upstream of this pathway, we also generated individual and dual deletions of the two FeS transfer proteins that deliver FeS clusters to Fd and downstream FeS proteins. We found that both transfer proteins are dispensable, but double deletion displayed a synthetic lethal phenotype, demonstrating their functional redundancy. These findings provide important insights into apicoplast biochemistry and drug development.

Project description:BackgroundDNA gyrase, an enzyme once thought to be unique to bacteria, is also found in some eukaryotic plastids including the apicoplast of Apicomplexa such as Plasmodium falciparum and Toxoplasma gondii which are important disease-causing organisms. DNA gyrase is an excellent target for antibacterial drugs, yet such antibacterials seem ineffective against Apicomplexa. Characterisation of the apicoplast gyrases would be a useful step towards understanding why this should be so. While purification of active apicoplast gyrase has proved impossible to date, in silico analyses have allowed us to discover differences in the apicoplast proteins. The resulting predicted structural and functional differences will be a first step towards development of apicoplast-gyrase specific inhibitors.ResultsWe have carried out sequence analysis and structural predictions of the enzymes from the two species and find that P. falciparum gyrase lacks a GyrA box, but T. gondii may retain one. All proteins contained signal/transport peptides for localization to the apicoplast but T. gondii Gyrase B protein lacks the expected hydrophobic region. The most significant difference is in the GyrA C-terminal domain: While the cores of the proteins, including DNA binding and cleavage regions are essentially unchanged, both apicoplast gyrase A proteins have C-terminal domains that are significantly larger than bacterial counterparts and are predicted to have different structures.ConclusionThe apicoplast gyrases differ significantly from bacterial gyrases while retaining similar core domains. T. gondii Gyrase B may have an unusual or inefficient mechanism of localisation to the apicoplast. P.falciparum gyrase, lacks a GyrA box and is therefore likely to be inefficient in DNA supercoiling. The C-terminal domains of both apicoplast Gyrase A proteins diverge significantly from the bacterial proteins. We predict that an additional structural element is present in the C-terminal domain of both apicoplast Gyrase A proteins, including the possibility of a β-pinwheel with a non-canonical number of blades. These differences undoubtedly will affect the DNA supercoiling mechanism and have perhaps evolved to compensate for the lack of Topoisomerase IV in the apicoplast. These data will be useful first step towards further characterisation and development of inhibitors for apicoplast gyrases.

Dataset Information

Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites.

Publications

Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites.

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