PMID:8662613

Clementz, T, Bednarski, JJ and Raetz, CR (1996) Function of the htrB high temperature requirement gene of Escherchia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate.J. Biol. Chem. 271:12095-102

Abstract

By assaying lysates of Escherichia coli generated with the hybrid lambda bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), we identified two clones (lambda232 and lambda233) capable of overexpressing the lauroyl transferase that functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). The E. coli DNA inserts in lambda232 and lambda233 suggested that a known gene (htrB) required for rapid growth above 33 degrees C might encode the lauroyl transferase. Using the intermediate (Kdo)2-lipid IVA as the laurate acceptor, extracts of strains with transposon insertions in htrB were found to contain no lauroyl transferase activity. Cells harboring hybrid htrB+ plasmids overproduced transferase activity 100-200-fold. The overproduced transferase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography. With lauroyl acyl carrier protein as the donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)2-lipid IVA. The rate of laurate incorporation was reduced by several orders of magnitude when either one or both Kdos were absent in the acceptor. With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or myristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow. Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltransferase of lipid A biosynthesis.

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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
LpxL		GO:0016740	transferase activity	IDA: Inferred from Direct Assay		F	LpxL is assayed using lauryl-ACP and Kdo2-[4’-32P]-tetraacyldisaccharide-1,4’ bisphosphate (Kdo2-[4’-32P]-lipid IVA) as substrates. The extent of the reaction is determined using a PhosphorImager after separation of the product from the substrates by thin layer chromatography (pg. 12096 and Figs. 2 and 3). The lauryl group is esterified to the hydroxyl group of the N-linked hydroxy-myristate of the distal glucosamine residue of Kdo2-[4’-32P]-lipid IVA (see Fig. 1). LpxL activity is not detected in strains with a lpxL::Tn10 insertion (Fig. 3 and Table II). The substrates are not commercially available but their preparation is described.	complete
LpxL		GO:0016020	membrane	IDA: Inferred from Direct Assay		C	LpxL is purified from membrane fractions obtained after disruption of overproducing strains by passage through a French pressure cell. Membranes were separated from the soluble fraction by centrifugation at 150,000 x g for 60 minutes (pg. 12096 and Table III).	complete
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