Characterisation of a putative glutamate 5‐kinase from Leishmania donovani

Natasha Sienkiewicz, Han Ong, Alan Fairlamb (Lead / Corresponding author)

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4 Citations (Scopus)
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Abstract

Previous metabolic studies have demonstrated that leishmania parasites are able to synthesise proline from glutamic acid and threonine from aspartic acid. The first committed step in both biosynthetic pathways involves an amino acid kinase, either a glutamate 5‐kinase (G5K; EC2.7.2.11) or an aspartokinase (EC2.7.2.4). Bioinformatic analysis of multiple leishmania genomes identifies a single amino acid‐kinase gene (LdBPK 262740.1) variously annotated as either a putative glutamate or aspartate kinase. To establish the catalytic function of this Leishmania donovani gene product, we have determined the physical and kinetic properties of the recombinant enzyme purified from Escherichia coli. The findings indicate that the enzyme is a bona fide G5K with no activity as an aspartokinase. Tetrameric G5K displays kinetic behaviour similar to its bacterial orthologues and is allosterically regulated by proline, the end product of the pathway. The structure‐activity relationships of proline analogues as inhibitors are broadly similar to the bacterial enzyme. However, unlike G5K from E. coli, leishmania G5K lacks a C‐terminal PUA (pseudouridine synthase and archaeosine transglycosylase) domain and does not undergo higher oligomerisation in the presence of proline. Gene replacement studies are suggestive, but not conclusive that G5K is essential.
Original languageEnglish
Pages (from-to)2662-2678
Number of pages17
JournalFEBS Journal
Volume285
Issue number14
Early online date19 May 2018
DOIs
Publication statusPublished - Jul 2018

Keywords

  • Leishmania
  • glutamate 5-kinase
  • RCWT
  • wild/type leishmania overexpressing G5K
  • SDR
  • single drug resistant line
  • DDR
  • double drug resistant line
  • PUA domain
  • pseudouridine synthase and archaeosine transglycosylase domain
  • P5CS
  • UTR
  • untranslated region
  • inhibitors
  • biochemical pathway
  • proline biosynthesis
  • drug target

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