Galactokinase-like protein from Leishmania donovani: Biochemical and structural characterization of a recombinant protein.

Baber, Hasana; Aghajani, Arega; Gallimore, B Harold; Bethel, Cassandra; Hyatt, James G; King, Elizabeth F B; Price, Helen P; Maciej-Hulme, Marissa L; Sari, Suat; Winter, Anja

Abstract

Leishmaniasis is a spectrum of conditions caused by infection with the protozoan Leishmania spp. parasites. Leishmaniasis is endemic in 98 countries around the world, and resistance to current anti-leishmanial drugs is rising. Our work has identified and characterised a previously unstudied galactokinase-like protein (GalK) in Leishmania donovani, which catalyses the MgATP-dependent phosphorylation of the C-1 hydroxyl group of d-galactose to galactose-1-phosphate. Here, we report the production of the catalytically active recombinant protein in E. coli, determination of its substrate specificity and kinetic constants, as well as analysis of its molecular envelope using in solution X-ray scattering. Our results reveal kinetic parameters in range with other galactokinases with an average apparent Km value of 76 μM for galactose, V and apparent K values with 4.46376 × 10  M/s and 0.021 s , respectively. Substantial substrate promiscuity was observed, with galactose being the preferred substrate, followed by mannose, fructose and GalNAc. LdGalK has a highly flexible protein structure suggestive of multiple conformational states in solution, which may be the key to its substrate promiscuity. Our data presents novel insights into the galactose salvaging pathway in Leishmania and positions this protein as a potential target for the development of pharmaceuticals seeking to interfere with parasite substrate metabolism. [Abstract copyright: Copyright © 2024. Published by Elsevier B.V.]

Citation

Baber, H., Aghajani, A., Gallimore, B. H., Bethel, C., Hyatt, J. G., King, E. F. B., …Winter, A. (2024). Galactokinase-like protein from Leishmania donovani: Biochemical and structural characterization of a recombinant protein. Biochimie, 223, 31-40. https://doi.org/10.1016/j.biochi.2024.03.017

• Journal Article Type: Article
• Acceptance Date: Mar 28, 2024
• Online Publication Date: Apr 4, 2024
• Publication Date: 2024-08
• Deposit Date: Apr 22, 2024
• Journal: Biochimie
• Print ISSN: 0300-9084
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 223
• Pages: 31-40
• DOI: https://doi.org/10.1016/j.biochi.2024.03.017
• Keywords: Leishmania donovani, Enzymatic activity, Small-angle scattering, Galactokinase