Crystallization and preliminary X-ray crystallographic studies of 3-deoxy-manno-octulosonate cytidylyltransferase from Haemophilus influenzae.
The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-manno-octulosonate (KDO) by forming CMP-KDO. It is essential for the biosynthesis of lipopolysaccharides in Gram-negative bacteria and is a potential target for the discovery of antibacterial agents. L-CKS from Haemophilus influenzae was overexpressed with a C-terminal hexahistidine tag in Escherichia coli and crystallized in the presence of the substrate KDO at 297 K using PEG 4000 as a precipitant and ethylene glycol as an additive. The diffraction limit and spot shape of the native crystal could be improved significantly by dehydration/annealing. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 83.1, c = 117.3 A. The presence of two monomers of recombinant L-CKS in the crystallographic asymmetric unit gives a reasonable V(M) of 2.05 A(3) Da(-1), with a solvent content of 40.0%.
Ku
Min-Je
MJ
Structural Proteomics Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, South Korea.
Yoon
Hye-Jin
HJ
Ahn
Hyung Jun
HJ
Kim
Hyung-Wook
HW
Baek
Seung-Hun
SH
Suh
Se Won
SW
D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism; D014867:Water / Q000737:chemistry
D017931:DNA Primers; D011994:Recombinant Proteins; D013400:Sugar Acids; D014867:Water; C002532:2-keto-3-deoxyoctonate; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase