Purification and characterization of the lipid A disaccharide synthase (LpxB) from Escherichia coli, a peripheral membrane protein.
Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2,3-diacylglucosamine 1-phosphate --> 2',3'-diacylglucosamine-(beta,1'-6)-2,3-diacylglucosamine 1-phosphate + UDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consists entirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purified E. coli and Haemophilus influenzae LpxB to near homogeneity on a 10-100 mg scale using protease-cleavable His(10)-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E. coli LpxB (M(r) approximately 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized with buffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide. Transmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxB is overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which no residual catalytic activity was detected. Our results provide a rational starting point for structural studies.
Metzger
Louis E
LE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Raetz
Christian R H
CR
D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006193:Haemophilus influenzae / Q000201:enzymology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D010447:Peptide Hydrolases / Q000378:metabolism; D012492:Salts / Q000737:chemistry
D001426:Bacterial Proteins; D008565:Membrane Proteins; D012492:Salts; D017351:N-Acetylglucosaminyltransferases; C050305:lipid A disaccharide synthase; D010447:Peptide Hydrolases