An alternative route for UDP-diacylglucosamine hydrolysis in bacterial lipid A biosynthesis.
The outer leaflet of the outer membranes of Gram-negative bacteria is composed primarily of lipid A, the hydrophobic anchor of lipopolysaccharide. Like Escherichia coli, most Gram-negative bacteria encode one copy of each of the nine genes required for lipid A biosynthesis. An important exception exists in the case of the fourth enzyme, LpxH, a peripheral membrane protein that hydrolyzes UDP-2,3-diacylglucosamine to form 2,3-diacylglucosamine 1-phosphate and UMP by catalyzing the attack of water at the alpha-P atom. Many Gram-negative organisms, including all alpha-proteobacteria and diverse environmental isolates, lack LpxH. Here, we report a distinct UDP-2,3-diacylglucosamine pyrophosphatase, designated LpxI, which has no sequence similarity to LpxH but generates the same products by a different route. LpxI was identified because its structural gene is located between lpxA and lpxB in Caulobacter crescentus. The lpxI gene rescues the conditional lethality of lpxH-deficient E. coli. Lysates of E. coli in which C. crescentus LpxI (CcLpxI) is overexpressed display high levels of UDP-2,3-diacylglucosamine pyrophosphatase activity. CcLpxI was purified to >90% homogeneity. CcLpxI is stimulated by divalent cations and is inhibited by EDTA. Unlike E. coli LpxH, CcLpxI is not inhibited by an increase in the concentration of detergent, and its pH dependency is different. When the CcLpxI reaction is conducted in the presence of H(2)(18)O, the (18)O is incorporated exclusively into the 2,3-diacylglucosamine 1-phosphate product, as judged by mass spectrometry, demonstrating that CcLpxI catalyzes the attack of water on the beta-P atom of UDP-2,3-diacylglucosamine.
Metzger
Louis E
LE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Raetz
Christian R H
CR
D016935:Caulobacter crescentus / Q000201:enzymology / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008050:Lipid A / Q000096:biosynthesis*; D053858:Metabolic Networks and Pathways; D011755:Pyrophosphatases / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D003902:Detergents; D008050:Lipid A; D014867:Water; D011755:Pyrophosphatases; C464799:UDP-2,3-diacylglucosamine pyrophosphatase