Overexpression, purification, crystallization and data collection on the Bordetella pertussis wlbD gene product, a putative UDP-GlcNAc 2'-epimerase.
The Boredetella pertussis wlbD gene product is a putative uridine-5-diphosphate N-acetylglucosamine (UDP-GlcNAc) 2'-epimerase involved in Band A lipopolysaccharide biosynthesis. The wlbD gene is homologous to Escherichia coli rffE (32% identical), an established UDP-GlcNAc 2'-epimerase that is involved in enterobacterial common antigen (ECA) formation. The structure of the rffE protein reveals an unexpected role for a bound sodium ion in orientating a substrate-binding alpha-helix in the enzyme active site. Whilst key active-site residues in rffE are present in the wlbD sequence, the sodium-binding residues outside the active site are absent. This raises questions about the modulation of enzyme activity in these two enzymes. The wlbD gene from B. pertussis has been cloned and overexpressed in E. coli and the resulting protein has been purified to homogeneity. In the current study, crystals of the mutant Gln339Arg wlbD enzyme have been obtained by sitting-drop vapour diffusion. Uncomplexed Gln339Arg and UDP-GlcNAc complex data sets have been collected in-house on a rotating-anode generator to 2.1 A. Combined, the data sets identify the space group as P2(1)2(1)2(1), with unit-cell parameters a = 78, b = 91, c = 125 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers and 53% solvent.
Sri Kannathasan
V
V
Centre for Carbohydrate Chemistry, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, England.
Staines
A G
AG
Dong
C J
CJ
Field
R A
RA
Preston
A G
AG
Maskell
D J
DJ
Naismith
J H
JH
D000942:Antigens, Bacterial / Q000737:chemistry; D001886:Bordetella pertussis / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; C083982:wecB protein, E coli