Characterization of Glucokinases from Pathogenic Free-Living Amoebae

Abstract

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent K_m values of 472.5 and 41.0 μM, while Homo sapiens Glck (HsGlck) has a value of 310 μM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent K_m values of 45.9 μM (AcGlck) and 124 μM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

Keywords: Acanthamoeba castellanii; Balamuthia mandrillaris; Naegleria fowleri; free-living amoeba; glucokinase; glycolysis; inhibitors; pentose phosphate pathway.

FIG 1

Effects of glucose and ATP on AcGlck and BmGlck activity. AcGlck or BmGlck (10 nM enzyme) activity was measured in response to increasing glucose concentrations (A and C) or ATP concentrations (B and D). For AcGlck, glucose (1 μM to 5.0 mM) and ATP (25 μM to 2.5 mM) were tested, while assays with BmGlck used concentrations of glucose from 25 μM to 10 mM and ATP from 72 μM to 5.0 mM. Enzymes were assayed in triplicate using a coupled reaction as described previously to measure enzyme activity (4), and standard deviations are indicated. (For some of the data points, the standard deviations are too small to be seen in the graph).

FIG 2

Effects of pH on AcGlck and BmGlck activity. Activity of AcGlck (A) and BmGlck (B) (both at 10 nM) was measured at different pH levels. Three different buffers with buffering capacity in the pH range tested were used for the assays: MES (morpholineethanesulfonic acid) sodium salt (pH 5.5 to 6.5), Tris-HCl (pH 7 to 9.0), and sodium borate (pH 9.5 to 10.0). Assays were carried out in triplicate, and standard deviations are indicated.

FIG 3

Effects of different substrates on AcGlck and BmGlck activity. (A) AcGlck and (B) BmGlck activity using various hexoses as substrates at 2 mM and 20 mM. The substrates tested were glucose (glc), mannose (man), fructose (fru), galactose (gal), and xylose (xyl). To test these, enzyme activity was measured in a reaction that included pyruvate kinase and lactate dehydrogenase as reporter enzymes. Reactions were carried out in triplicate, and standard deviations are indicated.

FIG 4

Impact of AMP and pyrophosphate on enzyme activity. The percent of (A) AcGlck (10 nM) and (B) BmGlck (10 nM) activity relative to activity scored from the standard reaction (which contains 1.5 mM ATP) as a consequence of inclusion of 5 mM AMP or pyrophosphate. Reactions were carried out in triplicate, and standard deviations are indicated. Significance was determined comparing results to the sample without additional agent added (No Add) using Welch’s t test (two tailed). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 5

Structural comparisons of amoebic Glcks. (A) Crystal structure of homodimeric BmGlck (ribbons; coloring by chain) bound to glucose (BGC). (B) Asymmetric unit and subunit structure of BmGlck (ribbons, with the small domain [residues 2 to 151 and 368 to 380] in light green and the large domain [residues 152 to 367] in orange) bound to glucose. (C) Glucose binding site of BmGlck. Dashed lines represent potential hydrogen bonds. Water molecules are shown as red spheres. (D) Comparison of protomeric structures of BmGlck and NfGlck. The crystal structures of BmGlck bound to glucose (light blue ribbons) and NfGlck bound to glucose and AMP-PNP (PDB entry 6DA0 [4]; pink ribbons) were superimposed using Chimera (24, 25). (E) Active-site comparison of amoebic Glcks. A molecular model of AcGlck (purple) was superimposed onto the crystal structure of NfGlck shown in panel D. The displayed glucose (green) is that bound to NfGlck. ATP (cyan) was modeled based on the conformation of AMP-PNP bound to human Glck (PDB entry 3ID8 [28]).