Abstract
Extracts of Escherichia coli contain an enzyme that generates the beta,1----6 linkage of lipid A from fatty-acylated monosaccharide precursors, according to the reaction: 2,3-diacyl-GlcN-1-P + UDP-2,3-diacyl-GlcN----2,3-diacyl-GlcN (beta, 1----6)2,3-diacyl-GlcN-1-P + UDP (Ray, B. L., Painter, G., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859). We now describe a membrane-bound kinase that phosphorylates the 4'-position of the above tetraacyldisaccharide 1-phosphate product. The lipid A 4'-kinase is distinct from the diglyceride kinase of E. coli. When crude membrane preparations are employed, several nucleoside triphosphates are able to support the phosphorylation of the tetraacyldisaccharide 1-phosphate, but ATP is the most efficient. The 4'-kinase requires Mg2+ and is stimulated by phospholipids, especially cardiolipin. Under optimal conditions the specific activity in crude extracts is 0.5 nmol/min/mg. The enzyme is rapidly inactivated by preincubation in the presence of detergents, such as Nonidet P-40 or octylglucoside, but phosphoenolpyruvate and glycerol stabilize the enzyme. The product generated in vitro has been characterized by fast atom bombardment mass spectrometry and by 1H and 31P NMR spectroscopy. Those analyses confirm that the 4' hydroxyl is the site of phosphorylation. The 4'-kinase reported here is likely to represent a key step in the de novo biosynthesis of lipid A.
Highlights
We describe a membrane-boundkinase that phosphorylates the 4‘-position of the above tetraacyldisaccharide 1phosphateproduct.Thelipid A 4”kinase is distinct fromthediglyceride kinase of E. coli
Diacylglucosamine 1-phosphate to serve as the precursor of the nonreducing end unitof lipid A (15) led us to identify an enzyme in extracts of wild-type cells capable of generating the p J - 6 linkage of lipid A by catalyzing the condensation of 2,3-diacylglucosamine 1-phosphateand UDP-2,3-diacylglucosamine ( X ), as shown in Fig. 1.This enzyme, termed the ationofthetetraacyldisaccharide1-phosphate, but lipid A disaccharide synthase, iscoded for by the lpzB
The kinase described in this article appears to be a novel enzyme that incorporates the 4'-monophosphate moiety of lipid A (Fig. 1).The existence of this kinase in extracts of E. coli supports the proposed biosynthetic scheme for lipid A
Summary
The sample was applied to a DEAE-cellulosecolumn (125-ml bedvolume) prepared in thesame solvent, andthe product was eluted as described by Raetz et al (22) for the purification of the precursor IVA. Membranes from wild-type cells (lanes[1] and 2) incorporate 32Pinto the glycerophospholipids (the more rapidly migrating components), independeach case the kinase product emerged at min 39, as did polar lipid ently of added tetraacyldisaccharide 1-phosphate. 60 mlof CHC13:methanol:water (2:3:1, v/v) was added, and the sample was applied to a second column (40-ml bed volume) of DEAE-cellulose in the same solvent (22) to remove the tetrabutylammonium phosphate. Fractions containingthe kinase product were adjusted by adding appropriate amounts of CHCI3, water, and concentrated HCl to generate a two-phase, acidic Bligh-Dyer system (11,22). Prolonged storage of the kinase product (or lipid X), as thefree acid, results in the gradual loss of the anomeric phosphate residue. Efficient conversion of the lipid substrate to product at a linear rate was observedonly if cardiolipin was included in the reaction mixture, together
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