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

Open AccessPublished:August 28, 2015DOI:https://doi.org/10.1074/jbc.M115.655100
      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 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-tRNAGln to Gln-tRNAGln 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.

      Introduction

      The human malaria parasite Plasmodium falciparum is responsible for 124–283 million cases of malaria and an estimated 0.6 million deaths every year (
      World Health Organization
      ). It contains a relict plastid, the remnant of an ancient secondary endosymbiotic event in which the eukaryotic progenitor of the malaria parasite engulfed a photosynthetic eukaryote known as the apicoplast (
      • Goodman C.D.
      • McFadden G.I.
      Targeting apicoplasts in malaria parasites.
      ). The Plasmodium apicoplast possesses a 35-kb circular genome with 60 genes (
      • Wilson R.J.
      • Denny P.W.
      • Preiser P.R.
      • Rangachari K.
      • Roberts K.
      • Roy A.
      • Whyte A.
      • Strath M.
      • Moore D.J.
      • Moore P.W.
      • Williamson D.H.
      Complete gene map of the plastid-like DNA of the malaria parasitePlasmodium falciparum.
      ) that encode components of the apicoplast transcriptional and translational apparatus such as RNA polymerase subunits, the elongation factor EF-Tu, several ribosomal proteins, rRNAs, and tRNAs (
      • Gardner M.J.
      • Feagin J.E.
      • Moore D.J.
      • Spencer D.F.
      • Gray M.W.
      • Williamson D.H.
      • Wilson R.J.
      Organisation and expression of small subunit ribosomal RNA genes encoded by a 35-kilobase circular DNA inPlasmodium falciparum.
      ,
      • Gardner M.J.
      • Williamson D.H.
      • Wilson R.J.
      A circular DNA in malaria parasites encodes an RNA polymerase like that of prokaryotes and chloroplasts.
      ,
      • Feagin J.E.
      • Werner E.
      • Gardner M.J.
      • Williamson D.H.
      • Wilson R.J.
      Homologies between the contiguous and fragmented rRNAs of the twoPlasmodium falciparum extrachromosomal DNAs are limited to core sequences.
      ,
      • Gardner M.J.
      • Preiser P.
      • Rangachari K.
      • Moore D.
      • Feagin J.E.
      • Williamson D.H.
      • Wilson R.J.
      Nine duplicated tRNA genes on the plastid-like DNA of the malaria parasitePlasmodium falciparum.
      ,
      • Preiser P.
      • Williamson D.H.
      • Wilson R.J.
      tRNA genes transcribed from the plastid-like DNA ofPlasmodium falciparum.
      ), as well as the SufB protein thought to play a role in FeS cluster formation (
      • Seeber F.
      Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists.
      ). Most apicoplast proteins, however, are encoded by the nuclear genome and are imported into the organelle post-translationally (
      • Waller R.F.
      • Reed M.B.
      • Cowman A.F.
      • McFadden G.I.
      Protein trafficking to the plastid ofPlasmodium falciparum is via the secretory pathway.
      ). Over 500 apicoplast-targeted proteins were identified in P. falciparum (
      • Gardner M.J.
      • Hall N.
      • Fung E.
      • White O.
      • Berriman M.
      • Hyman R.W.
      • Carlton J.M.
      • Pain A.
      • Nelson K.E.
      • Bowman S.
      • Paulsen I.T.
      • James K.
      • Eisen J.A.
      • Rutherford K.
      • Salzberg S.L.
      • et al.
      Genome sequence of the human malaria parasitePlasmodium falciparum.
      ,
      • Ralph S.A.
      • van Dooren G.G.
      • Waller R.F.
      • Crawford M.J.
      • Fraunholz M.J.
      • Foth B.J.
      • Tonkin C.J.
      • Roos D.S.
      • McFadden G.I.
      Tropical infectious diseases: metabolic maps and functions of thePlasmodium falciparum apicoplast.
      ), revealing apicoplast biosynthetic pathways for fatty acids (
      • Gardner M.J.
      • Tettelin H.
      • Carucci D.J.
      • Cummings L.M.
      • Aravind L.
      • Koonin E.V.
      • Shallom S.
      • Mason T.
      • Yu K.
      • Fujii C.
      • Pederson J.
      • Shen K.
      • Jing J.
      • Aston C.
      • Lai Z.
      • et al.
      Chromosome 2 sequence of the human malaria parasitePlasmodium falciparum.
      ,
      • Waller R.F.
      • Keeling P.J.
      • Donald R.G.
      • Striepen B.
      • Handman E.
      • Lang-Unnasch N.
      • Cowman A.F.
      • Besra G.S.
      • Roos D.S.
      • McFadden G.I.
      Nuclear-encoded proteins target to the plastid inToxoplasma gondii andPlasmodium falciparum.
      ), isoprenoid precursors (
      • Jomaa H.
      • Wiesner J.
      • Sanderbrand S.
      • Altincicek B.
      • Weidemeyer C.
      • Hintz M.
      • Türbachova I.
      • Eberl M.
      • Zeidler J.
      • Lichtenthaler H.K.
      • Soldati D.
      • Beck E.
      Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.
      ), and heme (
      • Nagaraj V.A.
      • Arumugam R.
      • Chandra N.R.
      • Prasad D.
      • Rangarajan P.N.
      • Padmanaban G.
      Localisation ofPlasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties.
      ), as well as enzymes for tRNA modification (
      • Ralph S.A.
      • van Dooren G.G.
      • Waller R.F.
      • Crawford M.J.
      • Fraunholz M.J.
      • Foth B.J.
      • Tonkin C.J.
      • Roos D.S.
      • McFadden G.I.
      Tropical infectious diseases: metabolic maps and functions of thePlasmodium falciparum apicoplast.
      ) and lipoylation (
      • Günther S.
      • Matuschewski K.
      • Müller S.
      Knockout studies reveal an important role ofPlasmodium lipoic acid protein ligase A1 for asexual blood stage parasite survival.
      ). Several of these pathways exhibit prokaryote-like features and are potential drug targets (
      • Ralph S.A.
      • van Dooren G.G.
      • Waller R.F.
      • Crawford M.J.
      • Fraunholz M.J.
      • Foth B.J.
      • Tonkin C.J.
      • Roos D.S.
      • McFadden G.I.
      Tropical infectious diseases: metabolic maps and functions of thePlasmodium falciparum apicoplast.
      ,
      • Jomaa H.
      • Wiesner J.
      • Sanderbrand S.
      • Altincicek B.
      • Weidemeyer C.
      • Hintz M.
      • Türbachova I.
      • Eberl M.
      • Zeidler J.
      • Lichtenthaler H.K.
      • Soldati D.
      • Beck E.
      Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.
      ,
      • Vaughan A.M.
      • O'Neill M.T.
      • Tarun A.S.
      • Camargo N.
      • Phuong T.M.
      • Aly A.S.
      • Cowman A.F.
      • Kappe S.H.
      Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.
      ). Recent studies have shown that apicoplast isoprenoid precursor biosynthesis is essential in P. falciparum asexual stages (
      • Yeh E.
      • DeRisi J.L.
      Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stagePlasmodium falciparum.
      ), indicating that the pathway cannot be bypassed by salvaging lipids from the host and may be a good drug target in asexual stages. The type II fatty acid and heme biosynthetic pathways, however, are not essential in the asexual stages (
      • Vaughan A.M.
      • O'Neill M.T.
      • Tarun A.S.
      • Camargo N.
      • Phuong T.M.
      • Aly A.S.
      • Cowman A.F.
      • Kappe S.H.
      Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.
      ), and although not good targets for asexual stage chemotherapy, they may prove to be valuable drug targets in liver stages (
      • Goodman C.D.
      • McFadden G.I.
      Targeting apicoplasts in malaria parasites.
      ).
      Translational accuracy is required to properly decipher the genetic code during protein synthesis. The fidelity of protein synthesis largely depends on the formation of correct aminoacyl-tRNAs by aminoacyl-tRNA synthetases (aaRSs).
      The abbreviations used are:
      aaRS
      aminoacyl-tRNA synthetase
      ACP
      acyl-carrier protein
      Glu-AdT
      glutamyl-tRNA amidotransferase
      GluRS
      glutamyl-tRNA synthetase
      GlnRS
      glutaminyl-tRNA synthetase
      Pb
      Plasmodium berghei
      Pf
      Plasmodium falciparum
      ND
      non-discriminating
      AdT
      amidotransferase
      AsnRS
      asparaginyl-tRNA synthetase
      AspRS
      aspartyl-tRNA synthetase
      Ni-NTA
      nickel-nitrilotriacetic acid.
      In the classic model, each species of aaRS strictly discriminates one amino acid from among the 20 canonical amino acids, as well as its cognate tRNA isoacceptor from the non-cognate tRNAs. However, genomic and biochemical analyses have revealed that the full complement of 20 aaRSs is used only in the eukaryotic cytoplasm and a minority of bacteria, whereas a majority of bacterial and archaeal genomes lack genes encoding glutaminyl-tRNA synthetase (GlnRS) and or asparaginyl-tRNA synthetase (AsnRS) (
      • Tumbula D.L.
      • Becker H.D.
      • Chang W.Z.
      • Söll D.
      Domain-specific recruitment of amide amino acids for protein synthesis.
      ). In these organisms, Gln-tRNAGln and/or Asn-tRNAAsn are synthesized via an indirect pathway (
      • Sheppard K.
      • Yuan J.
      • Hohn M.J.
      • Jester B.
      • Devine K.M.
      • Söll D.
      From one amino acid to another: tRNA-dependent amino acid biosynthesis.
      ). In most bacteria and archaea lacking GlnRS, tRNAGln is first misaminoacylated with Glu in a reaction catalyzed by a non-discriminating GluRS (ND-GluRS) that can glutamylate both tRNAGlu and tRNAGln (see Reactions 1 and 2).
      L glutamate + ATP + tRNA Gln Glu tRNA Gln + AMP + PP i
      Reaction 1


      L glutamine + ATP + Glu tRNA Gln Glu tRNA Gln + L glutamate + ADP + PP i
      Reaction 2


      The glutamyl residue of Glu-tRNAGln is then transamidated by a glutamyl-tRNAGln amidotransferase (Glu-AdT) in the presence of ATP using Gln as an amide donor, producing Gln-tRNAGln. Similarly, in the case of Asn-tRNAAsn formation in organisms lacking AsnRS, Asn-tRNAAsn is synthesized by a non-discriminating aspartyl-tRNA synthetase and an aspartyl-tRNAAsn amidotransferase (Asp-AdT). Two types of tRNA-dependent amidotransferases are known as follows: the heterotrimeric GatCAB (
      • Curnow A.W.
      • Hong Kw
      • Yuan R.
      • Kim Si
      • Martins O.
      • Winkler W.
      • Henkin T.M.
      • Söll D.
      Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.
      ) and the heterodimeric GatDE (
      • Tumbula D.L.
      • Becker H.D.
      • Chang W.Z.
      • Söll D.
      Domain-specific recruitment of amide amino acids for protein synthesis.
      ). Bacterial GatCAB functions as a Glu-AdT or an Asp-AdT in a species-specific manner. In some bacteria lacking both AsnRS and GlnRS, GatCAB acts as both a Glu-AdT and an Asp-AdT (
      • Sheppard K.
      • Yuan J.
      • Hohn M.J.
      • Jester B.
      • Devine K.M.
      • Söll D.
      From one amino acid to another: tRNA-dependent amino acid biosynthesis.
      ). As each subunit of GatCAB is encoded only in archaeal genomes lacking a gene for AsnRS, archaeal GatCAB seems to function as an Asp-AdT. GatDE is only found in archaea and functions as a Glu-AdT (
      • Sheppard K.
      • Yuan J.
      • Hohn M.J.
      • Jester B.
      • Devine K.M.
      • Söll D.
      From one amino acid to another: tRNA-dependent amino acid biosynthesis.
      ).
      Aminoacyl-tRNA formation is essential for protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how Plasmodium synthesizes Gln-tRNAGln in the apicoplast. The Plasmodium apicoplast genome does not encode any tRNA synthetases, and the nuclear genome does not contain an apicoplast-targeted GlnRS. We recently reported that a nucleus-encoded non-discriminating GluRS that is imported into the apicoplast is responsible for the formation of misacylated Glu-tRNAGln and is essential in the erythrocytic stages (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ). In this study, we aimed to further clarify the formation of Gln-tRNAGln in the Plasmodium apicoplast by a unique Plasmodium signature protein GatAB.

      Discussion

      To date, two different tRNA-dependent AdTs are known: the heterotrimeric GatCAB (
      • Curnow A.W.
      • Hong Kw
      • Yuan R.
      • Kim Si
      • Martins O.
      • Winkler W.
      • Henkin T.M.
      • Söll D.
      Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.
      ) and the heterodimeric GatDE (
      • Tumbula D.L.
      • Becker H.D.
      • Chang W.Z.
      • Söll D.
      Domain-specific recruitment of amide amino acids for protein synthesis.
      ) enzymes. The latter is an archaeal signature enzyme and serves as the Glu-AdT for Gln-tRNAGln biosynthesis in archaea (
      • Tumbula D.L.
      • Becker H.D.
      • Chang W.Z.
      • Söll D.
      Domain-specific recruitment of amide amino acids for protein synthesis.
      ). GatCAB is found in both bacteria and archaea (
      • Tumbula D.L.
      • Becker H.D.
      • Chang W.Z.
      • Söll D.
      Domain-specific recruitment of amide amino acids for protein synthesis.
      ,
      • Feng L.
      • Sheppard K.
      • Tumbula-Hansen D.
      • Söll D.
      Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu-tRNAGln kinase.
      ). In archaeal genomes, GatCAB is encoded only when an AsnRS is not (
      • Roy H.
      • Becker H.D.
      • Reinbolt J.
      • Kern D.
      When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism.
      ). All bacterial GatCAB enzymes studied to date are able to serve as both a Glu-AdT and an Asp-AdT in vitro (
      • Curnow A.W.
      • Tumbula D.L.
      • Pelaschier J.T.
      • Min B.
      • Söll D.
      Glutamyl-tRNA(Gln) amidotransferase inDeinococcus radiodurans may be confined to asparagine biosynthesis.
      ,
      • Becker H.D.
      • Min B.
      • Jacobi C.
      • Raczniak G.
      • Pelaschier J.
      • Roy H.
      • Klein S.
      • Kern D.
      • Söll D.
      The heterotrimericThermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln).
      ,
      • Raczniak G.
      • Becker H.D.
      • Min B.
      • Söll D.
      A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA inChlamydia trachomatis.
      ,
      • Salazar J.C.
      • Zúñiga R.
      • Raczniak G.
      • Becker H.
      • Söll D.
      • Orellana O.
      A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons inAcidithiobacillus ferrooxidans.
      ,
      • Cathopoulis T.J.
      • Chuawong P.
      • Hendrickson T.L.
      A thin-layer electrophoretic assay for Asp-tRNAAsn/Glu-tRNAGln amidotransferase.
      ,
      • Sheppard K.
      • Akochy P.M.
      • Salazar J.C.
      • Söll D.
      TheHelicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln.
      ). The activity/ies actually performed by bacterial GatCABs in vivo is/are determined by the non-discriminating aaRS (ND-GluRS and/or ND-AspRS) possessed by each organism. For example, bacteria such as B. subtilis (
      • Lapointe J.
      • Duplain L.
      • Proulx M.
      A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln inBacillus subtilis and efficiently misacylatesEscherichia coli tRNAGln1in vitro.
      ) that have an ND-GluRS but lack an ND-AspRS use their GatCAB solely as a Glu-AdT (
      • Curnow A.W.
      • Hong Kw
      • Yuan R.
      • Kim Si
      • Martins O.
      • Winkler W.
      • Henkin T.M.
      • Söll D.
      Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.
      ). In bacteria possessing an ND-AspRS but lacking an ND-GluRS (e.g. Pseudomonas aeruginosa, Neisseria meningitidis, Thermus thermophilus, and Deinococcus radiodurans), GatCAB serves only as an Asp-AdT (
      • Curnow A.W.
      • Tumbula D.L.
      • Pelaschier J.T.
      • Min B.
      • Söll D.
      Glutamyl-tRNA(Gln) amidotransferase inDeinococcus radiodurans may be confined to asparagine biosynthesis.
      ,
      • Becker H.D.
      • Min B.
      • Jacobi C.
      • Raczniak G.
      • Pelaschier J.
      • Roy H.
      • Klein S.
      • Kern D.
      • Söll D.
      The heterotrimericThermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln).
      ,
      • Becker H.D.
      • Reinbolt J.
      • Kreutzer R.
      • Giegé R.
      • Kern D.
      Existence of two distinct aspartyl-tRNA synthetases inThermus thermophilus. Structural and biochemical properties of the two enzymes.
      ,
      • Becker H.D.
      • Kern D.
      Thermus thermophilus: a link in evolution of the tRNA-dependent amino acid amidation pathways.
      ,
      • Becker H.D.
      • Roy H.
      • Moulinier L.
      • Mazauric M.H.
      • Keith G.
      • Kern D.
      Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase.
      ,
      • Min B.
      • Pelaschier J.T.
      • Graham D.E.
      • Tumbula-Hansen D.
      • Söll D.
      Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation.
      ,
      • Akochy P.M.
      • Bernard D.
      • Roy P.H.
      • Lapointe J.
      Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis inPseudomonas aeruginosa PAO1.
      ,
      • Bailly M.
      • Giannouli S.
      • Blaise M.
      • Stathopoulos C.
      • Kern D.
      • Becker H.D.
      A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine.
      ). In bacteria carrying both non-discriminating aaRSs (ND-GluRS and ND-AspRS) such as Chlamydia trachomatis (
      • Raczniak G.
      • Becker H.D.
      • Min B.
      • Söll D.
      A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA inChlamydia trachomatis.
      ) and H. pylori (
      • Salazar J.C.
      • Ahel I.
      • Orellana O.
      • Tumbula-Hansen D.
      • Krieger R.
      • Daniels L.
      • Söll D.
      Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates.
      ,
      • Skouloubris S.
      • Ribas de Pouplana L.
      • De Reuse H.
      • Hendrickson T.L.
      A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution.
      ,
      • Chuawong P.
      • Hendrickson T.L.
      The nondiscriminating aspartyl-tRNA synthetase fromHelicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity.
      ), GatCAB serves as a Glu/Asp-AdT (
      • Raczniak G.
      • Becker H.D.
      • Min B.
      • Söll D.
      A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA inChlamydia trachomatis.
      ,
      • Cathopoulis T.J.
      • Chuawong P.
      • Hendrickson T.L.
      A thin-layer electrophoretic assay for Asp-tRNAAsn/Glu-tRNAGln amidotransferase.
      ,
      • Sheppard K.
      • Akochy P.M.
      • Salazar J.C.
      • Söll D.
      TheHelicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln.
      ). The P. falciparum genome (
      • Gardner M.J.
      • Hall N.
      • Fung E.
      • White O.
      • Berriman M.
      • Hyman R.W.
      • Carlton J.M.
      • Pain A.
      • Nelson K.E.
      • Bowman S.
      • Paulsen I.T.
      • James K.
      • Eisen J.A.
      • Rutherford K.
      • Salzberg S.L.
      • et al.
      Genome sequence of the human malaria parasitePlasmodium falciparum.
      ) encodes two putative d-AspRS enzymes, one of which (PF3D7_0514300) possesses a predicted N-terminal apicoplast-targeting sequence, suggesting that it is imported into the apicoplast (
      • Foth B.J.
      • Ralph S.A.
      • Tonkin C.J.
      • Struck N.S.
      • Fraunholz M.
      • Roos D.S.
      • Cowman A.F.
      • McFadden G.I.
      Dissecting apicoplast targeting in the malaria parasitePlasmodium falciparum.
      ,
      • Zuegge J.
      • Ralph S.
      • Schmuker M.
      • McFadden G.I.
      • Schneider G.
      Deciphering apicoplast targeting signals–feature extraction from nuclear-encoded precursors ofPlasmodium falciparum apicoplast proteins.
      ). The other AspRS (PF3D7_0102900) lacks an apicoplast-targeting signal and is probably cytoplasmic (
      • Bhatt T.K.
      • Kapil C.
      • Khan S.
      • Jairajpuri M.A.
      • Sharma V.
      • Santoni D.
      • Silvestrini F.
      • Pizzi E.
      • Sharma A.
      A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasitePlasmodium falciparum.
      ,
      • Pino P.
      • Aeby E.
      • Foth B.J.
      • Sheiner L.
      • Soldati T.
      • Schneider A.
      • Soldati-Favre D.
      Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation inApicomplexa.
      ,
      • Bour T.
      • Akaddar A.
      • Lorber B.
      • Blais S.
      • Balg C.
      • Candolfi E.
      • Frugier M.
      Plasmodial aspartyl-tRNA synthetases and peculiarities inPlasmodium falciparum.
      ). Similarly, Plasmodium contains two putative AsnRSs as follows: one apicoplast-targeted (PF3D7_0509600) and the other cytoplasmic (PF3D7_0211800) (
      • Gardner M.J.
      • Hall N.
      • Fung E.
      • White O.
      • Berriman M.
      • Hyman R.W.
      • Carlton J.M.
      • Pain A.
      • Nelson K.E.
      • Bowman S.
      • Paulsen I.T.
      • James K.
      • Eisen J.A.
      • Rutherford K.
      • Salzberg S.L.
      • et al.
      Genome sequence of the human malaria parasitePlasmodium falciparum.
      ,
      • Gardner M.J.
      • Bishop R.
      • Shah T.
      • de Villiers E.P.
      • Carlton J.M.
      • Hall N.
      • Ren Q.
      • Paulsen I.T.
      • Pain A.
      • Berriman M.
      • Wilson R.J.
      • Sato S.
      • Ralph S.A.
      • Mann D.J.
      • Xiong Z.
      • et al.
      Genome sequence ofTheileria parva, a bovine pathogen that transforms lymphocytes.
      ,
      • Hall N.
      • Pain A.
      • Berriman M.
      • Churcher C.
      • Harris B.
      • Harris D.
      • Mungall K.
      • Bowman S.
      • Atkin R.
      • Baker S.
      • Barron A.
      • Brooks K.
      • Buckee C.O.
      • Burrows C.
      • Cherevach I.
      • et al.
      Sequence ofPlasmodium falciparum chromosomes 1, 3–9 and 13.
      ). Thus, in the cytoplasm and apicoplast of Plasmodium, Asn-tRNAAsn is formed via direct aminoacylation.
      We previously demonstrated that the P. falciparum nuclear genome (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ) encodes two putative GluRS enzymes. One, Pf3D7_1349200, appears to be cytoplasmic (
      • Pino P.
      • Aeby E.
      • Foth B.J.
      • Sheiner L.
      • Soldati T.
      • Schneider A.
      • Soldati-Favre D.
      Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation inApicomplexa.
      ). The other, Pf3D7_1357200, possesses a predicted N-terminal apicoplast-targeting sequence (
      • Foth B.J.
      • Ralph S.A.
      • Tonkin C.J.
      • Struck N.S.
      • Fraunholz M.
      • Roos D.S.
      • Cowman A.F.
      • McFadden G.I.
      Dissecting apicoplast targeting in the malaria parasitePlasmodium falciparum.
      ,
      • Zuegge J.
      • Ralph S.
      • Schmuker M.
      • McFadden G.I.
      • Schneider G.
      Deciphering apicoplast targeting signals–feature extraction from nuclear-encoded precursors ofPlasmodium falciparum apicoplast proteins.
      ), was localized to the apicoplast, and exhibits non-discriminating glutamylation activity in vitro, producing both Glu-tRNAGlu and Glu-tRNAGln. It is the first enzyme in the apicoplast's indirect aminoacylation pathway (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ). In this study we have further dissected the apicoplast's indirect aminoacylation pathway by identifying the PfGatA and PfGatB subunits of the apicoplast aminoacyl-tRNA AdT. Using episomal constructs in which GFP was fused to the predicted PfGatA and GatB apicoplast targeting sequences, we demonstrated that GFP was trafficked to the apicoplast in erythrocytic stage parasites. Minor overlaps between the anti-GFP and MitoTracker Red signals were observed where the apicoplast and mitochondrion appeared to contact one another (Fig. 3, C, panel ii, and D, panel ii), a phenomenon observed with other apicoplast-targeted proteins (
      • Hopkins J.
      • Fowler R.
      • Krishna S.
      • Wilson I.
      • Mitchell G.
      • Bannister L.
      The plastid inPlasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis.
      ,
      • van Dooren G.G.
      • Marti M.
      • Tonkin C.J.
      • Stimmler L.M.
      • Cowman A.F.
      • McFadden G.I.
      Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle ofPlasmodium falciparum.
      ). Furthermore, we myc-tagged the endogenous P. berghei chromosomal gatA gene, and we observed that the tagged protein was trafficked to the apicoplast, confirming the results obtained in P. falciparum with episomal constructs. Localization of P. falciparum GatAB solely in the apicoplast differs from the situation in Arabidopsis, where the GatCAB amidotransferase is targeted to both the plastid and the mitochondrion (
      • Duchêne A.M.
      • Giritch A.
      • Hoffmann B.
      • Cognat V.
      • Lancelin D.
      • Peeters N.M.
      • Zaepfel M.
      • Maréchal-Drouard L.
      • Small I.D.
      Dual targeting is the rule for organellar aminoacyl-tRNA synthetases inArabidopsis thaliana.
      ). Dual targeting of PfGatAB to the plastid and mitochondrion is also unlikely because, as in the related parasite T. gondii, the Plasmodium mitochondrion probably imports aminoacylated tRNAs from the cytoplasm (
      • Pino P.
      • Aeby E.
      • Foth B.J.
      • Sheiner L.
      • Soldati T.
      • Schneider A.
      • Soldati-Favre D.
      Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation inApicomplexa.
      ).
      We expressed recombinant PfGatA and PfGatB independently in E. coli (Fig. 6, A and B), and we combined them in vitro to examine their ability to transamidate apicoplast Glu-tRNAGlu or Glu-tRNAGln that had been previously glutamylated using recombinant PfGluRS (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ). PfGatAB demonstrated a remarkable tRNA substrate specificity by converting Glu-tRNAGln to Gln-tRNAGln but did not transamidate Glu-tRNAGlu (Fig. 6C). The Plasmodium apicoplast possesses an ND-GluRS (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ) but lacks an ND-AspRS (
      • Bhatt T.K.
      • Kapil C.
      • Khan S.
      • Jairajpuri M.A.
      • Sharma V.
      • Santoni D.
      • Silvestrini F.
      • Pizzi E.
      • Sharma A.
      A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasitePlasmodium falciparum.
      ,
      • Pino P.
      • Aeby E.
      • Foth B.J.
      • Sheiner L.
      • Soldati T.
      • Schneider A.
      • Soldati-Favre D.
      Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation inApicomplexa.
      ), and therefore it almost certainly utilizes the PfGatAB as a Glu-AdT. As in Plasmodium, bacteria such as B. subtilis (
      • Lapointe J.
      • Duplain L.
      • Proulx M.
      A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln inBacillus subtilis and efficiently misacylatesEscherichia coli tRNAGln1in vitro.
      ) that possess an ND-GluRS but lack an ND-AspRS use their GatCAB only as a Glu-AdT (
      • Curnow A.W.
      • Tumbula D.L.
      • Pelaschier J.T.
      • Min B.
      • Söll D.
      Glutamyl-tRNA(Gln) amidotransferase inDeinococcus radiodurans may be confined to asparagine biosynthesis.
      ). Because genes encoding GatA and GatB are present in all known Plasmodium genomes but not in any known organism in the other two domains, we concluded that GatAB is the Plasmodium glutamyl-tRNAGln amidotransferase (GatAB/Glu-AdT).
      We also showed that PfGluRS, PfGatA, and PfGatB, when briefly pre-mixed, can glutamylate apicoplast tRNAGln and transamidate it to form Gln-tRNAGln in vitro (Fig. 6D). This suggests that the Plasmodium apicoplast may contain a tRNAGln·ND-GluRS·GatA·GatB complex, akin to the transamidosomes described in archaea (
      • Rampias T.
      • Sheppard K.
      • Söll D.
      The archaeal transamidosome for RNA-dependent glutamine biosynthesis.
      ) and the eubacterium T. thermophilus (
      • Bailly M.
      • Blaise M.
      • Lorber B.
      • Becker H.D.
      • Kern D.
      The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis.
      ). Such a complex would prevent challenging the genetic code integrity as demonstrated for tRNA-dependent Asn formation (
      • Rampias T.
      • Sheppard K.
      • Söll D.
      The archaeal transamidosome for RNA-dependent glutamine biosynthesis.
      ).
      We used different approaches to test for the PfGatAB-catalyzed amidotransferase reaction. Our findings consistently showed that the Plasmodium parasite has established an evolved glutamyl-tRNA amidotransferase reaction that takes into account the absence of the gatC gene in the parasite genome and therefore does not require it for the transamidation reaction, contrary to the amidotransferase activity of the GatCAB paralog where the three subunits are required for enzyme activity (
      • Curnow A.W.
      • Hong Kw
      • Yuan R.
      • Kim Si
      • Martins O.
      • Winkler W.
      • Henkin T.M.
      • Söll D.
      Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.
      ). This is the first biochemical evidence for Glu-tRNAGln transamidation by a GatAB in the absence of a GatC subunit.
      We investigated the evolution of Plasmodium GatA and GatB subunits in comparison with the human GatA and GatB, the GatA and GatB subunits of bacterial GatCABs, the GatA and GatB subunits of other plastids, and the GatD and GatE subunits of archaeal GatDE. In the unrooted phylogeny of GatB and GatE proteins, Plasmodium GatBs were not placed within the bacterial GatB, archaeal GatE, or the plastid GatB clades but were placed on a separate branch (Fig. 3A). The divide is well supported with a bootstrap value of 100. In a similar fashion, the unrooted phylogeny of GatA and GatD proteins, Plasmodium GatA was not placed within the bacterial GatA, the plastid GatA, or the archaeal GatD clades but was placed on a separate branch (bootstrap value of 100) (Fig. 3B). Additionally the GatB and GatA sequences of T. parva and Babesia bovis, which like Plasmodium lack gatC in their genomes, were placed in the same clade as the Plasmodium orthologs. Taken in total, the results strongly suggest that GatAB, which is uniquely found in Apicomplexa that possess an apicoplast, is a paralog to GatCAB, GatFAB, and GatDE.
      To identify conserved and divergent features of PfGatB and PfGatA, homology modeling was performed in comparison with the known S. aureus GatCAB structure (Fig. 1) (
      • Nakamura A.
      • Yao M.
      • Chimnaronk S.
      • Sakai N.
      • Tanaka I.
      Ammonia channel couples glutaminase with transamidase reactions in GatCAB.
      ,
      • Nakamura A.
      • Sheppard K.
      • Yamane J.
      • Yao M.
      • Söll D.
      • Tanaka I.
      Two distinct regions inStaphylococcus aureus GatCAB guarantee accurate tRNA recognition.
      ). All functional features of bacterial GatCAB and archaeal GatDE are conserved in PfGatAB, but we found that PfGatAB had unique inserts that could not be fitted into the GatCAB model (Fig. 1). In archaeal GatDE, the unstructured N-terminal insert of GatD has been reported to play a structural role that gives it the ability to associate to GatE (
      • Schmitt E.
      • Panvert M.
      • Blanquet S.
      • Mechulam Y.
      Structural basis for tRNA-dependent amidotransferase function.
      ). GatF found only in fungal genomes (
      • Frechin M.
      • Senger B.
      • Brayé M.
      • Kern D.
      • Martin R.P.
      • Becker H.D.
      Yeast mitochondrial Gln-tRNA(Gln) is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS.
      ,
      • Araiso Y.
      • Huot J.L.
      • Sekiguchi T.
      • Frechin M.
      • Fischer F.
      • Enkler L.
      • Senger B.
      • Ishitani R.
      • Becker H.D.
      • Nureki O.
      Crystal structure ofSaccharomyces cerevisiae mitochondrial GatFAB reveals a novel subunit assembly in tRNA-dependent amidotransferases.
      ) and GatC found in bacterial, plant, and mammalian AdTs have similar unstructured characteristics and play a similar structural role where they reinforce the interaction between the GatA and GatB subunits by encircling their interface (
      • Nakamura A.
      • Yao M.
      • Chimnaronk S.
      • Sakai N.
      • Tanaka I.
      Ammonia channel couples glutaminase with transamidase reactions in GatCAB.
      ,
      • Nakamura A.
      • Sheppard K.
      • Yamane J.
      • Yao M.
      • Söll D.
      • Tanaka I.
      Two distinct regions inStaphylococcus aureus GatCAB guarantee accurate tRNA recognition.
      ). We were not able to identify a homolog of the gatC or gatF genes in the P. falciparum genome, leading us to speculate that the unique inserts present in PfGatAB could be playing a yet undetermined structural role of holding PfGatA and PfGatB together, similar to that of the insertion found in GatD. Crystallization studies on the PfGatAB enzyme will help determine the roles of these inserts in catalysis.
      Here, we have shown that besides possessing an apicoplast-targeted, non-discriminating GluRS (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ), malaria parasites contain a unique apicoplast-targeted glutamyl-tRNA amidotransferase that amidates Glu-tRNAGln in the absence of GatC. The apicoplast is an attractive drug target because it is essential to both blood and liver stage parasites (
      • Vaughan A.M.
      • O'Neill M.T.
      • Tarun A.S.
      • Camargo N.
      • Phuong T.M.
      • Aly A.S.
      • Cowman A.F.
      • Kappe S.H.
      Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.
      ,
      • Yeh E.
      • DeRisi J.L.
      Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stagePlasmodium falciparum.
      ), harbors several metabolic pathways absent in the host, and its transcriptional and translational machinery are of bacterial origin. The apicoplast's indirect aminoacylation pathway is probably essential in malaria parasites because the parasite genome does not encode an apicoplast-targeted GlnRS (
      • Bhatt T.K.
      • Kapil C.
      • Khan S.
      • Jairajpuri M.A.
      • Sharma V.
      • Santoni D.
      • Silvestrini F.
      • Pizzi E.
      • Sharma A.
      A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasitePlasmodium falciparum.
      ,
      • Pino P.
      • Aeby E.
      • Foth B.J.
      • Sheiner L.
      • Soldati T.
      • Schneider A.
      • Soldati-Favre D.
      Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation inApicomplexa.
      ,
      • Jackson K.E.
      • Habib S.
      • Frugier M.
      • Hoen R.
      • Khan S.
      • Pham J.S.
      • Ribas de Pouplana L.
      • Royo M.
      • Santos M.A.
      • Sharma A.
      • Ralph S.A.
      Protein translation inPlasmodium parasites.
      ). PfGluRS, the first enzyme in the apicoplast's indirect aminoacylation pathway, was refractory to gene deletion and is therefore essential for blood stage development (
      • Mailu B.M.
      • Ramasamay G.
      • Mudeppa D.G.
      • Li L.
      • Lindner S.E.
      • Peterson M.J.
      • DeRocher A.E.
      • Kappe S.H.
      • Rathod P.K.
      • Gardner M.J.
      A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
      ). In this study, the gene encoding for apicoplast PbGatA was refractory to gene deletion, despite the fact that it was accessible to myc tagging. Thus, both PfGluRS and PfGlu-AdT are potentially good drug targets.
      The emergence of resistant strains of P. falciparum continues to fuel an urgent need to develop new antimalarials. Besides our findings in this study, the Plasmodium GatAB amidotransferase has also been identified to be an important antimalarial drug target using in silico molecular modeling approaches (
      • Plaimas K.
      • Wang Y.
      • Rotimi S.O.
      • Olasehinde G.
      • Fatumo S.
      • Lanzer M.
      • Adebiyi E.
      • König R.
      Computational and experimental analysis identified 6-diazo-5-oxonorleucine as a potential agent for treating infection byPlasmodium falciparum.
      ). However, despite the chemical diversity of compound collections, robust inhibitors that act specifically against Plasmodium GatAB amidotransferases and not other amidotransferases have yet to be identified. Further investigations examining the requirements for apicoplast indirect aminoacylation across the Plasmodium life cycle and screening of compounds to identify specific inhibitors of plasmodial GluRS and Glu-AdT enzymes may lead to novel ways to target this pathway for chemotherapy.

      Author Contributions

      M. J. G. conceived and directed the project. M. J. G. and B. M. designed the research; B. M.M., K. F. W., K. B., G. R., L. L., J. A., and T. M. N. performed the experiments; and B. M. M., M. J. G., K. F. W., and K. B. wrote the manuscript. All authors approved the final version of the manuscript.

      Acknowledgments

      We thank Geoffrey McFadden for providing anti-ACP antibody, J. Lapointe and A. Weiner for plasmids expressing CCA-adding enzyme, and Y. M. Hou for the pGFIB vector. We thank MR4 for providing P. falciparum 3D7 parasites contributed by Dan Carucci and Alister Craig. The unpublished P. berghei genome sequence data were produced by the Pathogen Sequencing Group at the Wellcome Trust Sanger Institute and can be obtained on line. The unpublished C. velia and V. brassicaformis ortholog sequences were graciously provided by Arnab Pain and Hifzur Ansari of King Abdullah University of Science and Technology. The Chromera and Vitrella sequencing project was funded by a King Abdullah University of Science and Technology OCRF (GCR) award (FIC/2010/09) to Arnab Pain. We also thank Jonathan Eisen and Dongying Wu for their consulting on the phylogenetic analyses.

      References

        • World Health Organization
        World Malaria Report 2014. World Health Organization, Geneva, Switzerland2014
        • Goodman C.D.
        • McFadden G.I.
        Targeting apicoplasts in malaria parasites.
        Expert Opin. Ther. Targets. 2013; 17: 167-177
        • Wilson R.J.
        • Denny P.W.
        • Preiser P.R.
        • Rangachari K.
        • Roberts K.
        • Roy A.
        • Whyte A.
        • Strath M.
        • Moore D.J.
        • Moore P.W.
        • Williamson D.H.
        Complete gene map of the plastid-like DNA of the malaria parasitePlasmodium falciparum.
        J. Mol. Biol. 1996; 261: 155-172
        • Gardner M.J.
        • Feagin J.E.
        • Moore D.J.
        • Spencer D.F.
        • Gray M.W.
        • Williamson D.H.
        • Wilson R.J.
        Organisation and expression of small subunit ribosomal RNA genes encoded by a 35-kilobase circular DNA inPlasmodium falciparum.
        Mol. Biochem. Parasitol. 1991; 48: 77-88
        • Gardner M.J.
        • Williamson D.H.
        • Wilson R.J.
        A circular DNA in malaria parasites encodes an RNA polymerase like that of prokaryotes and chloroplasts.
        Mol. Biochem. Parasitol. 1991; 44: 115-123
        • Feagin J.E.
        • Werner E.
        • Gardner M.J.
        • Williamson D.H.
        • Wilson R.J.
        Homologies between the contiguous and fragmented rRNAs of the twoPlasmodium falciparum extrachromosomal DNAs are limited to core sequences.
        Nucleic Acids Res. 1992; 20: 879-887
        • Gardner M.J.
        • Preiser P.
        • Rangachari K.
        • Moore D.
        • Feagin J.E.
        • Williamson D.H.
        • Wilson R.J.
        Nine duplicated tRNA genes on the plastid-like DNA of the malaria parasitePlasmodium falciparum.
        Gene. 1994; 144: 307-308
        • Preiser P.
        • Williamson D.H.
        • Wilson R.J.
        tRNA genes transcribed from the plastid-like DNA ofPlasmodium falciparum.
        Nucleic Acids Res. 1995; 23: 4329-4336
        • Seeber F.
        Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists.
        Int. J. Parasitol. 2002; 32: 1207-1217
        • Waller R.F.
        • Reed M.B.
        • Cowman A.F.
        • McFadden G.I.
        Protein trafficking to the plastid ofPlasmodium falciparum is via the secretory pathway.
        EMBO J. 2000; 19: 1794-1802
        • Gardner M.J.
        • Hall N.
        • Fung E.
        • White O.
        • Berriman M.
        • Hyman R.W.
        • Carlton J.M.
        • Pain A.
        • Nelson K.E.
        • Bowman S.
        • Paulsen I.T.
        • James K.
        • Eisen J.A.
        • Rutherford K.
        • Salzberg S.L.
        • et al.
        Genome sequence of the human malaria parasitePlasmodium falciparum.
        Nature. 2002; 419: 498-511
        • Ralph S.A.
        • van Dooren G.G.
        • Waller R.F.
        • Crawford M.J.
        • Fraunholz M.J.
        • Foth B.J.
        • Tonkin C.J.
        • Roos D.S.
        • McFadden G.I.
        Tropical infectious diseases: metabolic maps and functions of thePlasmodium falciparum apicoplast.
        Nat. Rev. Microbiol. 2004; 2: 203-216
        • Gardner M.J.
        • Tettelin H.
        • Carucci D.J.
        • Cummings L.M.
        • Aravind L.
        • Koonin E.V.
        • Shallom S.
        • Mason T.
        • Yu K.
        • Fujii C.
        • Pederson J.
        • Shen K.
        • Jing J.
        • Aston C.
        • Lai Z.
        • et al.
        Chromosome 2 sequence of the human malaria parasitePlasmodium falciparum.
        Science. 1998; 282: 1126-1132
        • Waller R.F.
        • Keeling P.J.
        • Donald R.G.
        • Striepen B.
        • Handman E.
        • Lang-Unnasch N.
        • Cowman A.F.
        • Besra G.S.
        • Roos D.S.
        • McFadden G.I.
        Nuclear-encoded proteins target to the plastid inToxoplasma gondii andPlasmodium falciparum.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12352-12357
        • Jomaa H.
        • Wiesner J.
        • Sanderbrand S.
        • Altincicek B.
        • Weidemeyer C.
        • Hintz M.
        • Türbachova I.
        • Eberl M.
        • Zeidler J.
        • Lichtenthaler H.K.
        • Soldati D.
        • Beck E.
        Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.
        Science. 1999; 285: 1573-1576
        • Nagaraj V.A.
        • Arumugam R.
        • Chandra N.R.
        • Prasad D.
        • Rangarajan P.N.
        • Padmanaban G.
        Localisation ofPlasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties.
        Int. J. Parasitol. 2009; 39: 559-568
        • Günther S.
        • Matuschewski K.
        • Müller S.
        Knockout studies reveal an important role ofPlasmodium lipoic acid protein ligase A1 for asexual blood stage parasite survival.
        PLoS One. 2009; 4: e5510
        • Vaughan A.M.
        • O'Neill M.T.
        • Tarun A.S.
        • Camargo N.
        • Phuong T.M.
        • Aly A.S.
        • Cowman A.F.
        • Kappe S.H.
        Type II fatty acid synthesis is essential only for malaria parasite late liver stage development.
        Cell. Microbiol. 2009; 11: 506-520
        • Yeh E.
        • DeRisi J.L.
        Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stagePlasmodium falciparum.
        PLoS Biol. 2011; 9: e1001138
        • Tumbula D.L.
        • Becker H.D.
        • Chang W.Z.
        • Söll D.
        Domain-specific recruitment of amide amino acids for protein synthesis.
        Nature. 2000; 407: 106-110
        • Sheppard K.
        • Yuan J.
        • Hohn M.J.
        • Jester B.
        • Devine K.M.
        • Söll D.
        From one amino acid to another: tRNA-dependent amino acid biosynthesis.
        Nucleic Acids Res. 2008; 36: 1813-1825
        • Curnow A.W.
        • Hong Kw
        • Yuan R.
        • Kim Si
        • Martins O.
        • Winkler W.
        • Henkin T.M.
        • Söll D.
        Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 11819-11826
        • Mailu B.M.
        • Ramasamay G.
        • Mudeppa D.G.
        • Li L.
        • Lindner S.E.
        • Peterson M.J.
        • DeRocher A.E.
        • Kappe S.H.
        • Rathod P.K.
        • Gardner M.J.
        A nondiscriminating glutamyl-tRNA synthetase in thePlasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.
        J. Biol. Chem. 2013; 288: 32539-32552
        • Gardner M.J.
        • Bishop R.
        • Shah T.
        • de Villiers E.P.
        • Carlton J.M.
        • Hall N.
        • Ren Q.
        • Paulsen I.T.
        • Pain A.
        • Berriman M.
        • Wilson R.J.
        • Sato S.
        • Ralph S.A.
        • Mann D.J.
        • Xiong Z.
        • et al.
        Genome sequence ofTheileria parva, a bovine pathogen that transforms lymphocytes.
        Science. 2005; 309: 134-137
        • Hall N.
        • Pain A.
        • Berriman M.
        • Churcher C.
        • Harris B.
        • Harris D.
        • Mungall K.
        • Bowman S.
        • Atkin R.
        • Baker S.
        • Barron A.
        • Brooks K.
        • Buckee C.O.
        • Burrows C.
        • Cherevach I.
        • et al.
        Sequence ofPlasmodium falciparum chromosomes 1, 3–9 and 13.
        Nature. 2002; 419: 527-531
        • Aurrecoechea C.
        • Brestelli J.
        • Brunk B.P.
        • Dommer J.
        • Fischer S.
        • Gajria B.
        • Gao X.
        • Gingle A.
        • Grant G.
        • Harb O.S.
        • Heiges M.
        • Innamorato F.
        • Iodice J.
        • Kissinger J.C.
        • Kraemer E.
        • et al.
        PlasmoDB: a functional genomic database for malaria parasites.
        Nucleic Acids Res. 2009; 37: D539-D543
        • UniProt Consortium
        The Universal Protein Resource (UniProt) in 2010.
        Nucleic Acids Res. 2010; 38: D142-D148
        • Nakamura A.
        • Yao M.
        • Chimnaronk S.
        • Sakai N.
        • Tanaka I.
        Ammonia channel couples glutaminase with transamidase reactions in GatCAB.
        Science. 2006; 312: 1954-1958
        • Nakamura A.
        • Sheppard K.
        • Yamane J.
        • Yao M.
        • Söll D.
        • Tanaka I.
        Two distinct regions inStaphylococcus aureus GatCAB guarantee accurate tRNA recognition.
        Nucleic Acids Res. 2010; 38: 672-682
        • Arnold K.
        • Bordoli L.
        • Kopp J.
        • Schwede T.
        The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling.
        Bioinformatics. 2006; 22: 195-201
        • Schmitt E.
        • Panvert M.
        • Blanquet S.
        • Mechulam Y.
        Structural basis for tRNA-dependent amidotransferase function.
        Structure. 2005; 13: 1421-1433
        • Pettersen E.F.
        • Goddard T.D.
        • Huang C.C.
        • Couch G.S.
        • Greenblatt D.M.
        • Meng E.C.
        • Ferrin T.E.
        UCSF Chimera–a visualization system for exploratory research and analysis.
        J. Comput. Chem. 2004; 25: 1605-1612
        • Dereeper A.
        • Guignon V.
        • Blanc G.
        • Audic S.
        • Buffet S.
        • Chevenet F.
        • Dufayard J.F.
        • Guindon S.
        • Lefort V.
        • Lescot M.
        • Claverie J.M.
        • Gascuel O.
        Phylogeny.fr: robust phylogenetic analysis for the non-specialist.
        Nucleic Acids Res. 2008; 36: W465-W469
        • Thompson J.D.
        • Higgins D.G.
        • Gibson T.J.
        CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
        Nucleic Acids Res. 1994; 22: 4673-4680
        • Castresana J.
        Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis.
        Mol. Biol. Evol. 2000; 17: 540-552
        • Guindon S.
        • Gascuel O.
        A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood.
        Syst. Biol. 2003; 52: 696-704
        • Chevenet F.
        • Brun C.
        • Bañuls A.L.
        • Jacq B.
        • Christen R.
        TreeDyn: toward dynamic graphics and annotations for analyses of trees.
        BMC Bioinformatics. 2006; 7: 439
        • Trager W.
        • Jensen J.B.
        Human malaria parasites in continuous culture.
        Science. 1976; 193: 673-675
        • Tonkin C.J.
        • van Dooren G.G.
        • Spurck T.P.
        • Struck N.S.
        • Good R.T.
        • Handman E.
        • Cowman A.F.
        • McFadden G.I.
        Localization of organellar proteins inPlasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method.
        Mol. Biochem. Parasitol. 2004; 137: 13-21
        • Wu Y.
        • Sifri C.D.
        • Lei H.H.
        • Su X.Z.
        • Wellems T.E.
        Transfection ofPlasmodium falciparum within human red blood cells.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 973-977
        • Crabb B.S.
        • Cowman A.F.
        Characterization of promoters and stable transfection by homologous and nonhomologous recombination inPlasmodium falciparum.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7289-7294
        • Mikolajczak S.A.
        • Aly A.S.
        • Dumpit R.F.
        • Vaughan A.M.
        • Kappe S.H.
        An efficient strategy for gene targeting and phenotypic assessment in thePlasmodium yoelii rodent malaria model.
        Mol. Biochem. Parasitol. 2008; 158: 213-216
        • Labaied M.
        • Camargo N.
        • Kappe S.H.
        Depletion of thePlasmodium berghei thrombospondin-related sporozoite protein reveals a role in host cell entry by sporozoites.
        Mol. Biochem. Parasitol. 2007; 153: 158-166
        • Sheppard K.
        • Akochy P.M.
        • Söll D.
        Assays for transfer RNA-dependent amino acid biosynthesis.
        Methods. 2008; 44: 139-145
        • Pain A.
        • Renauld H.
        • Berriman M.
        • Murphy L.
        • Yeats C.A.
        • Weir W.
        • Kerhornou A.
        • Aslett M.
        • Bishop R.
        • Bouchier C.
        • Cochet M.
        • Coulson R.M.
        • Cronin A.
        • de Villiers E.P.
        • Fraser A.
        • et al.
        Genome of the host-cell transforming parasiteTheileria annulata compared withT. parva.
        Science. 2005; 309: 131-133
        • Frechin M.
        • Senger B.
        • Brayé M.
        • Kern D.
        • Martin R.P.
        • Becker H.D.
        Yeast mitochondrial Gln-tRNA(Gln) is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS.
        Genes Dev. 2009; 23: 1119-1130
        • Araiso Y.
        • Huot J.L.
        • Sekiguchi T.
        • Frechin M.
        • Fischer F.
        • Enkler L.
        • Senger B.
        • Ishitani R.
        • Becker H.D.
        • Nureki O.
        Crystal structure ofSaccharomyces cerevisiae mitochondrial GatFAB reveals a novel subunit assembly in tRNA-dependent amidotransferases.
        Nucleic Acids Res. 2014; 42: 6052-6063
        • Shin S.
        • Lee T.H.
        • Ha N.C.
        • Koo H.M.
        • Kim S.Y.
        • Lee H.S.
        • Kim Y.S.
        • Oh B.H.
        Structure of malonamidase E2 reveals a novel Ser-cisSer-Lys catalytic triad in a new serine hydrolase fold that is prevalent in nature.
        EMBO J. 2002; 21: 2509-2516
        • Harpel M.R.
        • Horiuchi K.Y.
        • Luo Y.
        • Shen L.
        • Jiang W.
        • Nelson D.J.
        • Rogers K.C.
        • Decicco C.P.
        • Copeland R.A.
        Mutagenesis and mechanism-based inhibition ofStreptococcus pyogenes Glu-tRNAGln amidotransferase implicate a serine-based glutaminase site.
        Biochemistry. 2002; 41: 6398-6407
        • Lamour V.
        • Quevillon S.
        • Diriong S.
        • N′Guyen V.C.
        • Lipinski M.
        • Mirande M.
        Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer.
        Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 8670-8674
        • Brown J.R.
        • Doolittle W.F.
        Gene descent, duplication, and horizontal transfer in the evolution of glutamyl- and glutaminyl-tRNA synthetases.
        J. Mol. Evol. 1999; 49: 485-495
        • Brinkmann H.
        • van der Giezen M.
        • Zhou Y.
        • Poncelin de Raucourt G.
        • Philippe H.
        An empirical assessment of long-branch attraction artefacts in deep eukaryotic phylogenomics.
        Syst. Biol. 2005; 54: 743-757
        • Martinsen E.S.
        • Perkins S.L.
        • Schall J.J.
        A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): evolution of life-history traits and host switches.
        Mol. Phylogenet. Evol. 2008; 47: 261-273
        • Arisue N.
        • Hashimoto T.
        • Mitsui H.
        • Palacpac N.M.
        • Kaneko A.
        • Kawai S.
        • Hasegawa M.
        • Tanabe K.
        • Horii T.
        ThePlasmodium apicoplast genome: conserved structure and close relationship ofP. ovale to rodent malaria parasites.
        Mol. Biol. Evol. 2012; 29: 2095-2099
        • Brayton K.A.
        • Lau A.O.
        • Herndon D.R.
        • Hannick L.
        • Kappmeyer L.S.
        • Berens S.J.
        • Bidwell S.L.
        • Brown W.C.
        • Crabtree J.
        • Fadrosh D.
        • Feldblum T.
        • Forberger H.A.
        • Haas B.J.
        • Howell J.M.
        • Khouri H.
        • et al.
        Genome sequence ofBabesia bovis and comparative analysis of apicomplexan hemoprotozoa.
        PLoS Pathog. 2007; 3: 1401-1413
        • Foth B.J.
        • Ralph S.A.
        • Tonkin C.J.
        • Struck N.S.
        • Fraunholz M.
        • Roos D.S.
        • Cowman A.F.
        • McFadden G.I.
        Dissecting apicoplast targeting in the malaria parasitePlasmodium falciparum.
        Science. 2003; 299: 705-708
        • Bhatt T.K.
        • Kapil C.
        • Khan S.
        • Jairajpuri M.A.
        • Sharma V.
        • Santoni D.
        • Silvestrini F.
        • Pizzi E.
        • Sharma A.
        A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasitePlasmodium falciparum.
        BMC Genomics. 2009; 10: 644
        • Pino P.
        • Aeby E.
        • Foth B.J.
        • Sheiner L.
        • Soldati T.
        • Schneider A.
        • Soldati-Favre D.
        Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA Met formylation inApicomplexa.
        Mol. Microbiol. 2010; 76: 706-718
        • Feng L.
        • Sheppard K.
        • Tumbula-Hansen D.
        • Söll D.
        Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu-tRNAGln kinase.
        J. Biol. Chem. 2005; 280: 8150-8155
        • Srivastava D.K.
        • Bernhard S.A.
        Metabolite transfer via enzyme-enzyme complexes.
        Science. 1986; 234: 1081-1086
        • Rampias T.
        • Sheppard K.
        • Söll D.
        The archaeal transamidosome for RNA-dependent glutamine biosynthesis.
        Nucleic Acids Res. 2010; 38: 5774-5783
        • Zuegge J.
        • Ralph S.
        • Schmuker M.
        • McFadden G.I.
        • Schneider G.
        Deciphering apicoplast targeting signals–feature extraction from nuclear-encoded precursors ofPlasmodium falciparum apicoplast proteins.
        Gene. 2001; 280: 19-26
        • Roy H.
        • Becker H.D.
        • Reinbolt J.
        • Kern D.
        When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9837-9842
        • Curnow A.W.
        • Tumbula D.L.
        • Pelaschier J.T.
        • Min B.
        • Söll D.
        Glutamyl-tRNA(Gln) amidotransferase inDeinococcus radiodurans may be confined to asparagine biosynthesis.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12838-12843
        • Becker H.D.
        • Min B.
        • Jacobi C.
        • Raczniak G.
        • Pelaschier J.
        • Roy H.
        • Klein S.
        • Kern D.
        • Söll D.
        The heterotrimericThermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln).
        FEBS Lett. 2000; 476: 140-144
        • Raczniak G.
        • Becker H.D.
        • Min B.
        • Söll D.
        A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA inChlamydia trachomatis.
        J. Biol. Chem. 2001; 276: 45862-45867
        • Salazar J.C.
        • Zúñiga R.
        • Raczniak G.
        • Becker H.
        • Söll D.
        • Orellana O.
        A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons inAcidithiobacillus ferrooxidans.
        FEBS Lett. 2001; 500: 129-131
        • Cathopoulis T.J.
        • Chuawong P.
        • Hendrickson T.L.
        A thin-layer electrophoretic assay for Asp-tRNAAsn/Glu-tRNAGln amidotransferase.
        Anal. Biochem. 2007; 360: 151-153
        • Sheppard K.
        • Akochy P.M.
        • Salazar J.C.
        • Söll D.
        TheHelicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln.
        J. Biol. Chem. 2007; 282: 11866-11873
        • Lapointe J.
        • Duplain L.
        • Proulx M.
        A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln inBacillus subtilis and efficiently misacylatesEscherichia coli tRNAGln1in vitro.
        J. Bacteriol. 1986; 165: 88-93
        • Becker H.D.
        • Reinbolt J.
        • Kreutzer R.
        • Giegé R.
        • Kern D.
        Existence of two distinct aspartyl-tRNA synthetases inThermus thermophilus. Structural and biochemical properties of the two enzymes.
        Biochemistry. 1997; 36: 8785-8797
        • Becker H.D.
        • Kern D.
        Thermus thermophilus: a link in evolution of the tRNA-dependent amino acid amidation pathways.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12832-12837
        • Becker H.D.
        • Roy H.
        • Moulinier L.
        • Mazauric M.H.
        • Keith G.
        • Kern D.
        Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase.
        Biochemistry. 2000; 39: 3216-3230
        • Min B.
        • Pelaschier J.T.
        • Graham D.E.
        • Tumbula-Hansen D.
        • Söll D.
        Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 2678-2683
        • Akochy P.M.
        • Bernard D.
        • Roy P.H.
        • Lapointe J.
        Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis inPseudomonas aeruginosa PAO1.
        J. Bacteriol. 2004; 186: 767-776
        • Bailly M.
        • Giannouli S.
        • Blaise M.
        • Stathopoulos C.
        • Kern D.
        • Becker H.D.
        A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine.
        Nucleic Acids Res. 2006; 34: 6083-6094
        • Salazar J.C.
        • Ahel I.
        • Orellana O.
        • Tumbula-Hansen D.
        • Krieger R.
        • Daniels L.
        • Söll D.
        Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13863-13868
        • Skouloubris S.
        • Ribas de Pouplana L.
        • De Reuse H.
        • Hendrickson T.L.
        A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 11297-11302
        • Chuawong P.
        • Hendrickson T.L.
        The nondiscriminating aspartyl-tRNA synthetase fromHelicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity.
        Biochemistry. 2006; 45: 8079-8087
        • Bour T.
        • Akaddar A.
        • Lorber B.
        • Blais S.
        • Balg C.
        • Candolfi E.
        • Frugier M.
        Plasmodial aspartyl-tRNA synthetases and peculiarities inPlasmodium falciparum.
        J. Biol. Chem. 2009; 284: 18893-18903
        • Hopkins J.
        • Fowler R.
        • Krishna S.
        • Wilson I.
        • Mitchell G.
        • Bannister L.
        The plastid inPlasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis.
        Protist. 1999; 150: 283-295
        • van Dooren G.G.
        • Marti M.
        • Tonkin C.J.
        • Stimmler L.M.
        • Cowman A.F.
        • McFadden G.I.
        Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle ofPlasmodium falciparum.
        Mol. Microbiol. 2005; 57: 405-419
        • Duchêne A.M.
        • Giritch A.
        • Hoffmann B.
        • Cognat V.
        • Lancelin D.
        • Peeters N.M.
        • Zaepfel M.
        • Maréchal-Drouard L.
        • Small I.D.
        Dual targeting is the rule for organellar aminoacyl-tRNA synthetases inArabidopsis thaliana.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 16484-16489
        • Bailly M.
        • Blaise M.
        • Lorber B.
        • Becker H.D.
        • Kern D.
        The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis.
        Mol. Cell. 2007; 28: 228-239
        • Jackson K.E.
        • Habib S.
        • Frugier M.
        • Hoen R.
        • Khan S.
        • Pham J.S.
        • Ribas de Pouplana L.
        • Royo M.
        • Santos M.A.
        • Sharma A.
        • Ralph S.A.
        Protein translation inPlasmodium parasites.
        Trends Parasitol. 2011; 27: 467-476
        • Plaimas K.
        • Wang Y.
        • Rotimi S.O.
        • Olasehinde G.
        • Fatumo S.
        • Lanzer M.
        • Adebiyi E.
        • König R.
        Computational and experimental analysis identified 6-diazo-5-oxonorleucine as a potential agent for treating infection byPlasmodium falciparum.
        Infect. Genet. Evol. 2013; 20: 389-395