Publications

• Characterisation of a putative glutamate 5-kinase from Leishmania donovani.

  Sienkiewicz N., Ong H.B., Fairlamb A.H.

  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 aspar ... >> More

  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.<h4>Enzymes</h4>Glutamate 5-kinase (EC2.7.2.11); aspartokinase (EC2.7.2.4). << Less

  FEBS J. 285:2662-2678(2018) [PubMed] [EuropePMC]

• Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis.

  Smith C.J., Deutch A.H., Rushlow K.E.

  gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalt ... >> More

  gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline. << Less

  J. Bacteriol. 157:545-551(1984) [PubMed] [EuropePMC]