Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin.
The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
Hye-Jin
HJ
Yoon
Min-Je
MJ
Ku
Hyung Jun
HJ
Ahn
Byung Il
BI
Lee
Bunzo
B
Mikami
Se Won
SW
Suh
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D005578:Fosfomycin / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000201:enzymology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*
D005578:Fosfomycin; D014537:Uridine Diphosphate N-Acetylglucosamine; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase