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PMID:19883124

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Citation

Metzger, LE 4th and Raetz, CR (2009) Purification and characterization of the lipid A disaccharide synthase (LpxB) from Escherichia coli, a peripheral membrane protein. Biochemistry 48:11559-71

Abstract

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.

Links

PubMed PMC2787792 Online version:10.1021/bi901750f

Keywords

Bacterial Proteins/metabolism; Catalysis; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Haemophilus influenzae/enzymology; Mass Spectrometry; Membrane Proteins/metabolism; Microscopy, Electron, Transmission; N-Acetylglucosaminyltransferases/chemistry; N-Acetylglucosaminyltransferases/isolation & purification; N-Acetylglucosaminyltransferases/metabolism; Osmolar Concentration; Peptide Hydrolases/metabolism; Salts/chemistry

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:LPXB

located_in

GO:0019897: extrinsic component of plasma membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ECOLI:LPXB

enables

GO:0005543: phospholipid binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:LPXB

part_of

GO:0019897: extrinsic component of plasma membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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