Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium.
Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in D-mannose metabolism and supply of the activated mannose donor guanosine diphosphate D-mannose (GDP-D-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni-NTA affinity column chromatography. Purified MPI crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 A. A data set extending to 1.66 A resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.
Gowda
Giri
G
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Sagurthi
Someswar Rao
SR
Savithri
H S
HS
Murthy
M R N
MR
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000201:enzymology*
D017931:DNA Primers; D008359:Mannose-6-Phosphate Isomerase