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<p>Gram-negative bacteria possess an asymmetric outer membrane in which the inner
leaflet is composed primarily of phospholipids while the outer leaflet contains both
phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects
Gram-negative bacteria from antibiotics and other environmental stressors. The lipid
A anchor of LPS is a glucosamine-based saccharolipid that is further modified with
core and O-antigen sugars. In addition to serving a structural role as the hydrophobic
anchor of LPS, lipid A is recognized by the innate immune system in animal cells and
macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria;
in most species, a single copy of each bio-synthetic gene is present. The exception
is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine
hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate
and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives
lack an lpxH orthologue, yet these species must possess an activity analogous to that
of LpxH. We used bioinformatics approaches to identify a candidate gene, designated
lpxI, encoding this activity in the model organism Caulobacter crescentus. We then
demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we
have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase
activity in vitro. We have developed high-yield purification schema for Caulobacter
crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI
and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI,
which has no known homologues, consists of two novel domains connected by a linker.
Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate
in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant
to 3.0 Å; preliminary comparison with the product-complexed model reveals striking
differences. The findings described herein set the stage for further mechanistic and
structural characterization of this novel enzyme.</p>
<p></p>
<p>In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic
gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus
influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli
LpxB activity is dependent upon the bulk surface concentration of its substrates in
a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface.
E. coli LpxB partitions with membranes, but this interaction is partially abolished
in high-salt conditions, suggesting that a significant component of LpxB's membrane
association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane
protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by
autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme
of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular
membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected
conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic
activity is detected. Our data support the hypothesis that LpxB performs catalysis
at the cytoplasmic surface of the inner membrane, and provide a rational starting-point
for structural studies. This work contributes to knowledge of the small but growing
set of structurally and mechanistically characterized enzymes which perform chemistry
upon lipids.</p>
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