Expression, purification, crystallization and preliminary characterization of uridine 5'-diphospho-N-acetylmuramoyl L-alanyl-D-glutamate:lysine ligase (MurE) from Streptococcus pneumoniae 110K/70.
An ORF designated sp1530 (murE) in the Streptococcus pneumoniae TIGR4 genome sequence, identified as uridine 5'-diphospho-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE; EC 6.3.2.7), was cloned into the high-expression plasmid pET21b and overexpressed in Escherichia coli BL21 (DE3) Star. The enzyme was purified in three steps to 99% purity. Crystals were obtained by the hanging-drop vapour-diffusion method at 291 K from solutions containing 25%(w/v) polyethylene glycol 2000 monomethylether, 0.2 M potassium thiocyanate, 0.1 M MES pH 6.5 in the presence of uridine 5'-diphospho-N-acetylmuramoyl alanyl glutamate (UDP-MurNAc-L-Ala-D-Glu) with and without 5'-adenylyl imidophosphate (AMP-PNP), a non-hydrolysable analogue of ATP. Diffraction data to 1.5 and 2.7 A, respectively, were collected at the European Synchrotron Radiation Facility (ESRF). Crystals grown in the presence of two ligands belong to space group P1, with unit-cell parameters a = 68.4, b = 71.4, c = 74.8 A, alpha = 73.4, beta = 80.5, gamma = 72.3 degrees. Crystals grown in the presence of UDP-MurNAc-L-Ala-D-Glu alone belong to space group P2(1), with unit-cell parameters a = 71.1, b = 129.4, c = 74.6 A, beta = 106.3 degrees.
Blewett
Anne M
AM
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England.
Lloyd
Adrian J
AJ
Echalier
Aude
A
Fülöp
Vilmos
V
Dowson
Christopher G
CG
Bugg
Timothy D H
TD
Roper
David I
DI
D000818:Animals; D002498:Centrifugation; D002645:Chickens; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008024:Ligands; D008025:Ligases / Q000737:chemistry* / Q000302:isolation & purification; D008956:Models, Chemical; D010457:Peptidoglycan / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013010:Sonication; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D017356:Synchrotrons
D008024:Ligands; D010457:Peptidoglycan; D008025:Ligases; C491820:uridine 5'-diphospho-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase