Function of the htrB high temperature requirement gene of Escherichia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate.
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.
Clementz
T
T
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Bednarski
J J
JJ
Raetz
C R
CR
D000215:Acylation; D000217:Acyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins; D010582:Bacteriophage lambda / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007850:Lauric Acids / Q000378:metabolism*; D008050:Lipid A / Q000096:biosynthesis*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007850:Lauric Acids; D008050:Lipid A; C030358:lauric acid; D000217:Acyltransferases; C492237:LpxL protein, E coli; C118473:LpxL protein, bacteria