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-diphosp hate N-acetylglucosamine (UDP-GlcNAc) 2'-epimerase involved in Band A lipop olysaccharide biosynthesis. The wlbD gene is homologous to Escherichia coli rffE (32% identical), an established UDP-GlcNAc 2'-epimerase that is invol ved in enterobacterial common antigen (ECA) formation. The structure of the rffE protein reveals an unexpected role for a bound sodium ion in orientat ing 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-b inding residues outside the active site are absent. This raises questions a bout 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 resu lting protein has been purified to homogeneity. In the current study, cryst als of the mutant Gln339Arg wlbD enzyme have been obtained by sitting-drop vapour diffusion. Uncomplexed Gln339Arg and UDP-GlcNAc complex data sets ha ve been collected in-house on a rotating-anode generator to 2.1 Angstrom. C ombined, the data sets identify the space group as P2(1)2(1)2(1), with unit -cell parameters a = 78, b = 91, c = 125 Angstrom, alpha = beta = gamma = 9 0 degrees. The asymmetric unit contains two monomers and 53% solvent.