Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

Abstract

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the N. fowleri genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (Homo sapiens Glck), suggesting that parasite-specific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent K_m values of 42.5 ± 7.3 μM and 141.6 ± 9.9 μM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 μM that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.

Keywords: Naegleria fowleri; glucokinase.

FIG 1

(A) N. fowleri trophozoites were seeded at 2 × 10⁶ cells/ml in control medium (Nelson’s complete medium) and cultured with or without glucose. (B) Cells (1 × 10⁶ cells/ml) were incubated with or without 3-bromopyruvate (50 mM) for 2 days prior to imaging. Images were captured on an EVOS imaging system, and data are representative of multiple experiments. Scale bar, 20 μM.

FIG 2

Effects of ATP, glucose, and pH on NfGlck activity. (A) NfGlck (10 nM) activity in response to increasing glucose concentrations (2.44 to 312 μM) (A), increasing ATP concentrations (0.04 to 5.0 mM) (B), and different pH levels (C). Two different buffers with buffering capacity in the range of pH tested were used for the assay, Tris-Cl (pH 7 to 8.5) and sodium carbonate (pH 9.0 to 10.5). For pH analysis, glucose and ATP concentrations were 425 μM and 1.5 mM ATP, respectively. Reactions were carried out in triplicate, and standard deviations are indicated.

FIG 3

(A) NfGlck (10 nM) activity with various hexoses (5 mM). (B) Impact of phosphoryl-bearing substrates on NfGlck activity. NfGlck activity was measured in the presence of 1.5 mM ATP supplemented with ADP, PP_i, and ATP (all at 5 mM). Assays were carried out in triplicate, and standard deviations are indicated.

FIG 4

Structure of a crystallographic dimer of NfGlck bound to AMPPNP (ANP) and glucose (Glu) is shown in two color schemes. The left monomer is colored by secondary structure elements. The right monomer shows the smaller α/β domain (Pro23-Asn171 and Asn384-Leu400) in lavender and the larger α/β domain (His174-Glu382) in light blue. The glucose molecule is mostly buried, while part of AMPPNP is solvent accessible.

FIG 5

Nucleotide and glucose binding sites of various Glcks. (A) In NfGlck, AMPPNP (ANP) and glucose (Glu) are bound with a strong hydrogen bond network, including hydrogen bonds from the 1- and 2-hydroxyl groups to His231. AMPPNP and glucose are shown in sticks, while residues and water molecules interacting with the ligands are shown in thick bonds. Residues Lys367′, His369′, and Trp370 are from the other molecule in the crystallographic dimer. (B) In TcGlck, ADP and glucose are bound with a very similar hydrogen bond pattern. Ser210 replaces His231 and cannot engage in a hydrogen bond with glucose. Residues Met334′, Phe337′, and Phe339′ are located in the other chain of the dimer. (C) TcGlck bound to several glucosamine ligands (BENZ-GLCN, green, PDB entry 5BRD; CBZ-GLCN, gold, PDB entry 5BRE; HPOP-GLCN, orange, PDB entry 5BRF; BDT-GLCN, brown, PDB entry 5BRH), the amine moiety is part of the space that the His231 side chain of NfGlck occupies.