Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogenTrypanosoma cruziin complex with substrate

Abstract

An N-terminally truncated version of the enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi lacking the first 37 residues was crystallized both in its apo form and in a binary complex with glucose 6-phosphate. The crystals both belonged to space group P2 1 and diffracted to 2.85 and 3.35 Å resolution, respectively. Self-rotation function maps were consistent with point group 222. The structure was solved by molecular replacement, confirming a tetrameric quaternary structure.

The structure of a Trypanosoma cruzi glucose‐6‐phosphate dehydrogenase reveals differences from the mammalian enzyme

“…S1A,B). Size exclusion chromatography data indicate that Tc ΔG6PDH forms a tetramer, in agreement with the previous work . When Tc G6PDH‐L is expressed in E. coli , almost all the protein formed inclusion bodies .…”

“…After a 6‐year gap, Ortíz et al . described the crystallisation of a short form of Tc G6PDH ( Tc G6PDH‐S; Met38 to Ala555) and made available the coordinates of an apo (PDB entry: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4E9I, at 2.85 Å resolution) and an enzyme‐substrate complex (PDB entry: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4EM5, 3.35 Å resolution). More recently, the Seattle Structural Genomic Centre for Infectious Diseases deposited the structure of Ma G6PDH at 2.3 Å resolution .…”

Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi

“…The crystal structure of apo- (PDB 4E9I) and G6P-bound Tc G6PDH (PDB 4EM5) have recently been obtained [15] and a full structural characterization will be reported elsewhere. Tc G6PDH is a tetrameric protein with each subunit composed of an N-terminal domain with a typical Rossmann-fold involved in NADP + binding and a C-terminal domain that contains the G6P binding site and residues engaged in subunit interactions [15]. The catalytic site of G6PDH localizes at the interface between the N- and C-terminal domains and far from regions participating in protein dimerization and tetramerization [17,18,19].…”

“…The crystal structure of a truncated form of Tc G6PDH lacking the first 37 amino acids and co-crystallized in presence of its physiological substrate G6P (PDB ID: 4EM5; resolution 3.35 Å) [15] was pretreated by means of the Protein Preparation Wizard tool of the Maestro suite 9.2 [26] at default settings, adding hydrogen atoms, deleting water molecules, fill in missing amino acids side chains and eventual missing loop using Prime [27], generating ionization states of the co-crystallized ligand at pH 7 ± 3.0 using Epik [28] and checking amino acid protonation state using Propka, always at physiological pH [29]. NADP + , EA and all the other steroid structures discussed here were modeled with LigPrep [30] at default settings.…”

“…Tc G6PDH in its apo- and substrate-bound form has been recently crystallized by our group and the corresponding structures solved (PDB 4E9I and PDB 4EM5, respectively) [15]. Here we exploit the structural information available for Tc G6PDH to address the binding mode of steroids to the enzyme.…”