Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli.
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.
Milla
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ME
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Raetz
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CR
D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008050:Lipid A / Q000096:biosynthesis; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017421:Sequence Analysis
D008050:Lipid A; D010455:Peptides; D007544:Isopropyl Thiogalactoside; D005993:Glycerolphosphate Dehydrogenase; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase; D017351:N-Acetylglucosaminyltransferases; C050305:lipid A disaccharide synthase