Ribokinase from Leishmania donovani: Purification, characterization and X-ray crystallographic analysis

Abstract

Leishmania is an auxotrophic protozoan parasite which acquires d-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating d-ribose using ATP to produce d-ribose-5-phosphate. To understand its structure and function, the gene encoding RK from L. donovani was cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed the Tm to be 317.2 K. Kinetic parameters were obtained by functional characterization of L. donovani RK, and the Km values for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 μM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space group P61, with unit-cell parameters a = b = 100.25, c = 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da-1 and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.Ribokinase from L. donovani was cloned, purified, characterized and crystallized, and data were collected to a resolution of 1.95 Å. X-ray crystallographic analysis showed the presence of two molecules in the asymmetric unit, and human ribokinase could be used as a template to determine the three-dimensional structure.