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; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/11.html) or an aspartokinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/4.html). 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.EnzymesGlutamate 5‐kinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/11.html); aspartokinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/4.html).
Highlights
Leishmania parasites are the causative agents of visceral, cutaneous and mucocutaneous leishmaniasis
This study provides definitive evidence that the protein encoded by LdBPK_262740.1 is a bone fide glutamate 5-kinase (G5K) (EC 2.7.2.11) and not an aspartate kinase (EC 2.7.2.4)
The kinetic parameters are broadly equivalent to the few prokaryotic G5Ks that have been studied in any detail (Table 1)
Summary
Leishmania parasites are the causative agents of visceral, cutaneous and mucocutaneous leishmaniasis. The life-threatening visceral form is responsible for 30 000 to 50 000 deaths per annum and the incidence of the disfiguring cutaneous form is estimated at 700 000 to 1 300 000 new cases per annum (https://www.dndi.org/ diseases-projects/leishmaniasis/). The disease is transmitted between mammalian hosts by the bite of a female sand fly. During their life cycle, the parasites undergo remarkable morphological and biochemical changes, ranging from motile flagellated promastigote forms living in the alkaline midgut of the sand-fly vector to sessile amastigotes growing in the acidic parasitophorous vacuole of the host macrophage [4]. In addition to intermittent blood feeds, the female sand fly feeds on sugars (mainly sucrose) either from aphid honeydew or from plants
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