Crystallization and preliminary X-ray characterization of theglpX-encoded class II fructose-1,6-bisphosphatase fromMycobacterium tuberculosis

Abstract

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7 Å and belonged to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 131.3, c = 143.2 Å . The structure has been solved by molecular replacement and is currently undergoing refinement.

Current Trends and Advances in Bulk Crystallization and Freeze-Drying of Biopharmaceuticals

“…4) [16]. It is important to note here that while larger crystals were easily obtained, the edges and surface properties of larger crystals were ill-defined (Fig.…”

“…Tuberculosis Structural Genomics Consortium (TBSGC), formed as a part of the PSI is one such consortium dedicated towards solving structures of proteins from the pathogen Mycobacterium tuberculosis ( Mtb) the causative agent of tuberculosis (TB). Novel purification and crystallization means and crystal structures for Mtb proteins, as reported by consortium members and nonmembers have provided a meaningful insight into drug discovery and design efforts towards countering TB [2,10,11,15,16,32,38]. Figure 3 describes a typical process flow of structural elucidation for proteins by X-ray crystallography [44].…”

Metabolomics of Central Carbon Metabolism in Mycobacterium tuberculosis

“…3.1.3.11) is a key enzyme of gluconeogenesis that catalyzes the dephopshorylation of fructose-1,6-bisphosphate (FBP) to fructose-6-phosphate. The M. tuberculosis genome is annotated to encode a single type II, metal-ion-dependent FBPase, GlpX (Rv1099c), whose structure and activity have been characterized in purified recombinant form, with kinetic parameters similar to those of other type II FBPases (31,32). Though not essential for in vitro growth on glycolytic carbon sources, insertional transposon mutagenesis studies have implicated glpX as being essential for in vivo growth in a mouse model of tuberculosis (5,23,24).…”

Crystal structure ofActinobacillus pleuropneumoniaeHMW1C glycosyltransferase

“…We have previously reported the cloning, expression and purification to homogeneity of the purified enzyme and present the initial biochemical characterization [3]. MtFBPase displayed Michaelis-Menten kinetics for the substrate fructose 1, 6-bisphosphate.…”