Introduction Human pathogenic Leishmania protozoa comprise d

Introduction Human pathogenic Leishmania protozoa comprise 20 different species that are distributed throughout the world and cause the disease leishmaniasis [1]. Transmission primarily occurs through the bite of a female sand fly and clinical manifestations of the disease span from cutaneous lesions to visceral infections [2]. The parasitic protozoa utilize d-glucose as a major preferential carbon source to support their cellular growth and survival [3]. Amino acids and fatty acids can act as alternative carbon sources when d-glucose levels are limited [4]; however, all cultured stages of Leishmania exhibit severe growth restraints under such conditions [3, 5]. d-Glucose is also another important metabolic source for the generation of NADPH through the PPP, where NADPH has a role in serving as a reducing agent in maintaining redox balance and in producing precursors for DNA and RNA biosynthesis [6]. Based on the utility of d-glucose for Leishmania proliferation, metabolic pathways that include this monosaccharide as a substrate can be the subject of inhibition for leishmaniasis drug discovery. Enzymes of the glycolytic and PPP metabolisms have been studied as potential drug targets for the treatment of trypanosomatid diseases that include Chagas’ disease, Human African Sleeping Sickness, and leishmaniasis [[7], [8], [9]]. Up to the present time, there has been a lack of investigations on the Leishmania glucose kinases (i.e. hexokinase or glucokinase) as potential drug targets for the treatment of leishmaniasis. The glucose kinases are transferase enzymes responsible in the conversion of substrate d-glucose and co-substrate ATP to yield products G6P and ADP, where a Mg2+ ion is required for catalysis [10]. These enzymes are principally distinguished by their amino Calcium Gluceptate 1° structure and molecular weight range, which was thoroughly described by Kawai and colleagues [11]. Leishmania parasites have a hexokinase [12, 13] and a glucokinase [14] that are localized in the glycosome [15]. This is different than human cells, which contain four isoenzymes of hexokinase, belonging to the hexokinase group, designated HsHxK IIV. Furthermore, the inhibition of a glucose kinase from Leishmania may prove to be an important drug discovery strategy. The work herein focused on the X-ray crystal structure determination of glucokinase from the species L. braziliensis (LbGlcK) along with a kinetic characterization. Accordingly, active site structural comparisons to the previously solved structures of T. cruzi glucokinase (TcGlcK) [16] and HsHxKIV [17] will aid in the understanding of whether LbGlcK can be inhibited with monosaccharide-based competitive inhibitors in a selective manner.
Materials and methods
Results and discussion We report for His6-LbGlcK a KM (d-glucose) of 6.61 ± 2.63 mM, a kcat (d-glucose) of 3.59 × 103 ± 0.72 × 103 min−1, a kcat/KM (d-glucose) of 5.71 × 102 ± 1.08 × 102 mM−1 min−1, and a KM (ATP) of 0.338 ± 0.080 mM (see Figure S1 in SI for Michaelis-Menten plots). The LbGlcK KM (d-glucose) is close to the previously reported L. major glucokinase KM (d-glucose) of 3.30 mM and the KM values with respect to ATP are essentially identical (e.g. KM (ATP) of 0.35 mM for L. major glucokinase) [14]. Table S1 in SI shows a comparison between kinetic parameters of recombinant glucokinases from selected trypanosomatid species. Briefly, the KM values (with respect to d-glucose) for the listed Leishmania spp. glucokinases are slightly higher when compared to T. cruzi glucokinase (KM of 1.00 mM) and the KM with respect to ATP is principally the same for all of these glucokinases. The cDNA construct used in this study produced a truncation mutant of LbGlcK that excluded a 13-residue segment of the C-terminal region (residues: KSADQVVPTKSHL). We decided to use such a construct design because the analysis of the LbGlcK amino acid full-length sequence using the program DISOPRED3 [20] suggested that this segment, in particular, was disordered and could thereby hinder crystallization. The truncation created for full-length LbGlcK compared similarly to the truncation that was previously employed for that of full-length TcGlcK (a 9-residue C-terminal amino acid exclusion) [16]. In that, full-length TcGlcK also revealed the same disordered region at the C-terminus. Moreover, although we did not assay the full-length LbGlcK enzyme, we did not expect that the removal of its C-terminal segment would make any difference in enzymatic assay properties. This can be explained by the comparison to the results observed for TcGlcK, in which the full-length protein [14] and its truncation mutant (exclusion of a 9-residue C-terminal segment: VGKKQKAQL) [16] were previously assayed. The assays in the two studies revealed the same KM value for the substrate d-glucose, which was KM = 1 mM (see Table S1 in the Supplementary Section). In terms of the possibility of changes in structural folding due to the LbGlcK truncation, we did not expect such an issue since disorder defined by Ward and colleagues [20] is a protein segment that is flexible and dynamic that usually extends partially or fully into bulk solvent.