Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase fromEscherichia coli

Abstract

Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5 K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19 mg ml À1 FBPA I in 0.1 M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0 Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9 Å , = 124.6 . The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48 Å 3 Da À1 and a solvent content of 50.5%.

Screening and Identification of Antigenic Proteins from the Hard Tick <i>Dermacentor silvarum</i> (Acari: Ixodidae)

“…As we all know, FBA is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate [ 23 ]. FBA was also identified as an immunogenic protein in Streptococcus pneumoniae .…”

ESBL Escherichia coli Isolates Have Enhanced Gut Colonization Capacity Compared to Non-ESBL Strains in Neonatal Mice

“…This tool compares pathways across genomes using KEGG pathway maps. Several crucial genes tended to have higher copy numbers in the ESBL strains, such as 6-phospho-beta-glucosidase (hydrolyzes a variety of glucosides [ 33 ]), fructose bisphosphatase (important for regulation of gluconeogenesis [ 34 ]), fructose-biphosphate aldolase (central regulator in glycolysis and gluconeogenesis pathways [ 35 ]), fumarate hydratase (key contributor to E. coli fitness under iron limitation [ 36 ]), and NADH:ubiquinone reductase (central regulator of both the aerobic and anaerobic respiratory chain [ 41 ]). Two ESBL E. coli strains (ST28 and ST410) also had significantly higher copy number of metabolic genes than all other strains, including non-ESBL strains.…”

Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid

“…Then, the crystals were flash cooled under a nitrogen stream. Diffraction data were collected at the BL17U/18U/19U beamlines of the Shanghai Synchrotron Radiation Facility (Shanghai, China) [ 26 ]. X-ray diffraction data were indexed, integrated, and scaled with HKL-2000 [ 27 ] or HKL-3000 [ 28 ].…”