PMID:8982270
| Citation |
Milla, ME and Raetz, CR (1996) Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli. Biochim. Biophys. Acta 1304:245-53 |
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| Abstract |
Variants of the Escherichia coli UDP-GlcNAc O-acyltransferase (LpxA) and of the lipid A disaccharide synthase (LpxB) containing affinity chromatography tags (C-terminal histidine 8 [H8] tails) were constructed in order to investigate whether or not these enzymes interact with other E. coli proteins. These variants (LpxA-H8 and LpxB-H8) had specific activities in vitro that were similar to wild-type enzymes. Crude extracts made from E. coli cells expressing LpxA-H8 or LpxB-H8 were chromatographed over Ni(2+)-NTA-Agarose, and proteins purifying with the tagged proteins were identified by SDS-PAGE, followed by blotting and N-terminal microsequencing. At high levels of LpxB-H8 expression, two heat-shock proteins (DnaK and GroEL) were associated with the disaccharide synthase, but not with the acyltransferase. Another major protein recovered with LpxB-H8 (both at low and high levels of expression) was the aerobic glycerol-3-phosphate dehydrogenase (GlpD). The latter interaction was specific, since GlpD did not bind the affinity resin when the affinity tag was present on the UDP-GlcNAc O-acyltransferase (LpxA-H8). Velocity centrifugation experiments indicated that both wild-type LpxB and GlpD sedimented together under some conditions, but these aggregates were smaller than and distinct from inner membranes. Our findings suggest a possible new mechanism by which the biosynthetic pathways for lipid A and glycerophospholipids may be coordinated. |
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| Keywords |
Acyltransferases/metabolism; Amino Acid Sequence; Centrifugation, Density Gradient; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/metabolism; Gene Expression/genetics; Glycerolphosphate Dehydrogenase/metabolism; Isopropyl Thiogalactoside/pharmacology; Lipid A/biosynthesis; Molecular Sequence Data; N-Acetylglucosaminyltransferases/metabolism; Peptides/chemistry; Peptides/metabolism; Plasmids/genetics; Protein Binding; Sequence Analysis |
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