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Biochimie ; 223: 31-40, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38579894

RESUMO

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, Vmax and apparent Kcat values with 4.46376 × 10-9 M/s and 0.021 s-1, 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.


Assuntos
Leishmania donovani , Proteínas de Protozoários , Proteínas Recombinantes , Leishmania donovani/enzimologia , Leishmania donovani/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Especificidade por Substrato , Galactoquinase/metabolismo , Galactoquinase/genética , Galactoquinase/química , Cinética , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/metabolismo
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