An alternative route for UDP-diacylglucosamine hydrolysis in bacterial lipid a biosynthesis

Article abstract of DOI:10.1021/bi1008744

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 α-P atom. Many Gram-negative organisms, including all α-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 H₂¹⁸O, the ¹⁸O 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 β-P atom of UDP-2,3-diacylglucosamine.

Full text of DOI:10.1021/bi1008744

Biochemistry 2010, 49, 6715–6726 6715
DOI: 10.1021/bi1008744
An Alternative Route for UDP-Diacylglucosamine Hydrolysis in
†
Bacterial Lipid A Biosynthesis
Louis E. Metzger IV and Christian R. H. Raetz*
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received May 30, 2010; Revised Manuscript Received July 4, 2010
ABSTRACT: 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 R-P atom. Many
Gram-negative organisms, including all R-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
1
8
18
is different. When the CcLpxI reaction is conducted in the presence of H2 O, the O is incorporated
exclusively into the 2,3-diacylglucosamine 1-phosphate product, as judged by mass spectrometry, demon-
strating that CcLpxI catalyzes the attack of water on the β-P atom of UDP-2,3-diacylglucosamine.
Gram-negative bacteria possess inner and outer membranes.
The outer membrane is asymmetric, having a periplasm-facing
leaflet comprised of phospholipids, and an outer leaflet consisting
primarily of lipid A, the hydrophobic anchor of lipopolysacchar-
EcLpxH products, UMP and 2,3-diacylglucosamine 1-phosphate
(lipid X), generated from UDP-2,3-diacylglucosamine in the pre-
sence of H2 O, revealed that this hydrolase catalyzes the attack
1
8
of water at the R-P atom of UDP-2,3-diacylglucosamine with
1
18
ide (LPS) (1, 2). The biosynthesis of lipid A is required for
incorporation of the O isotope into the UMP product (8)
growth and viability in most Gram-negative bacteria (1, 3).
Therefore, the conserved, constitutive enzymes of lipid A bio-
synthesis (Scheme 1) are excellent targets against which to
develop new antibiotics (4-6). This study focuses on the catalysis
of UDP-2,3-diacylglucosamine hydrolysis (7, 8), the fourth step
of the pathway (Scheme 1).
(Scheme 2).
Most Gram-negative bacteria contain one gene encoding each
enzyme of the lipid A biosynthetic pathway (Scheme 1) (3). These
genes are easily identified by sequence comparisons (9). However,
LpxH is a notable exception. Many bacterial species, including all
R-proteobacteria, many δ-proteobacteria, all spirochetes, all
cyanobacteria, and diverse environmental strains, such as the
hyperthermophile Aquifex aeolicus, lack LpxH orthologues.
They nevertheless produce lipid A using the same enzymes
upstream and downstream of UDP-2,3-diacylglucosamine, as
demonstrated for Rhizobium leguminosarum and Rhizobium etli,
which lack LpxH (10, 11). These findings suggest that alternative
enzyme(s) catalyzing UDP-2,3-diacylglucosamine hydrolysis
must exist in these bacteria to provide the appropriate substrates
for LpxB (Scheme 1).
While a lack of sequence homology hampers the search for
the genes encoding these alternative hydrolases, genomic
context can sometimes assist in the identification of novel
gene functions (12-14). In this work, we identify and char-
acterize LpxI, a protein unrelated to LpxH that catalyzes
UDP-2,3-diacylglucosamine hydrolysis by attack of water
on the β-P atom instead of the R-P atom (Scheme 2). The
gene encoding LpxI was identified because of its location
between lpxA and lpxB in certain R-proteobacteria, such as
Caulobacter crescentus and Mesorhizobium loti, which lack
The lpxH gene encodes the essential UDP-2,3-diacylglucosa-
mine hydrolase of Escherichia coli (7, 8). E. coli LpxH (EcLpxH)
and its orthologues are distantly related to a large superfamily of
metallo-phosphoesterases with diverse functions. EcLpxH is a
peripheral membrane enzyme that displays apparent surface
dilution kinetics (8), and it requires divalent cations, preferably
2þ
2þ
31
Mn , but not Mg , for activity (8). P NMR analysis of the
†
This research was supported by National Institutes of Health Grant
GM-51310 to C.R.H.R. and LIPID MAPS Large Scale Collaborative
Grant GM-069338. L.E.M. was supported by National Science Foundation
Graduate Research Fellowship 2005029353.
*To whom correspondence should be addressed. Phone: (919) 684-
3
384. Fax: (919) 684-8885. E-mail: raetz@biochem.duke.edu.
Abbreviations: BCA, bicinchoninic acid; DTT, dithiothreitol; dUT-
1
Pase, dUTP diphosphatase; ESI-MS, electrospray ionization mass
spectrometry; FPLC, fast protein liquid chromatography; GlcN, glu-
cosamine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
IPTG, isopropyl β-D-1-thiogalactopyranoside; LC, liquid chromatog-
raphy; lipid X, 2,3-diacylglucosamine 1-phosphate; LPS, lipopolysac-
charide; PAGE, polyacrylamide gel electrophoresis; PAP, purple acid
phosphatase; PBS, phosphate-buffered saline; PCR, polymerase chain
reaction; TLC, thin layer chromatography.
r 2010 American Chemical Society
Published on Web 07/07/2010
pubs.acs.org/Biochemistry

6
716 Biochemistry, Vol. 49, No. 31, 2010
Metzger and Raetz
a
Scheme 1: Role of UDP-Diacylglucosamine Pyrophosphatases in Lipid A Biosynthesis
a
LpxH is primarily found in γ- and β-proteobacteria, whereas LpxI is present in R-proteobacteria, many δ-proteobacteria, and some
environmental isolates, as described in detail in Table 2 of the Supporting Information. Chlamydia and Cyanobacteria contain neither LpxH
nor LpxI but do possess LpxD and LpxB. Protein sequence data can be found at http://www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi.
a
Scheme 2: LpxH and LpxI Catalyze UDP-Diacylglucosamine Hydrolysis by Different Mechanisms
a
Oxygen from the water attacking the R-phosphate in the LpxH-catalyzed reaction is colored red, whereas oxygen from the water
attacking the β-phosphate in the LpxI-catalyzed reaction is colored magenta.
LpxH (Figure 1) (15, 16). The different hydrolytic mechanism
employed by LpxI explains why LpxI and LpxH share no
overall sequence similarity or even any conserved active site
residues (Figure 2). LpxI belongs to a unique protein super-
family (DUF1009) (17) for which no biochemical functions
have been previously reported.

Article
Biochemistry, Vol. 49, No. 31, 2010 6717
F
IGURE 1: Comparison of the lpxD-fabZ-lpxA-lpxB gene clusters of E. coli, C. crescentus, and M. loti. Arrows represent open reading frames and
their directions of transcription. A gene of unknown function encoding a protein designated as DUF2009 is situated between lpxA and lpxB in
C. cresentus and M. loti and also is present in many other bacteria, like A. aeolicus (not shown), that lack LpxH (15, 16, 32). The DUF1009
orthologues, CC1910 and MLL0631, are highlighted with a red box. Gene lengths are not drawn exactly to scale.
MATERIALS AND METHODS
The restriction endonucelases XbaI and SalI were used to digest
these plasmids, liberating linear E. coli lpxH or C. crescentus lpxI,
Chemicals and Reagents. Yeast extract, tryptone, and Bacto
agar were purchased from Difco (Detroit, MI). Glass-backed silica
gel 60 thin layer chromatography (TLC) plates were from EMD
Chemicals (Darmstadt, Germany). Chloroform, pyridine, metha-
nol, and acetic acid were from EMD Science (Gibbstown, NJ).
0
0
each flanked by 5 Xba and 3 SalI restriction sites, and containing
a pET21b-encoded ribosome binding site 7 bp upstream from the
start codon. These segments were then ligated, as described
above, into appropriately digested pBAD30 or pBAD33 plas-
mids (18). The resulting plasmids are listed in Table 1.
32
γ- P was purchased from PerkinElmer (Waltham, MA). The
i
Construction of E. coli lpxH Deletion Strains. A deletion
mutant of E. coli LpxH was generated with an in-frame kana-
mycin (Kan) insertion/substitution. Primers KanFlank_FW and
KanFlank_RV (see Table 1 of the Supporting Information) were
used to amplify a Kan cassette from plasmid pET28b (Novagen/
EMD Chemicals). These primers were designed with 40 bp
overhangs complementary to the regions of the E. coli chromo-
some immediately flanking lpxH. KanFlank_FW also added a
4
-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
phosphate-buffered saline components, HCl, and salts were
purchased from Sigma-Aldrich (St. Louis, MO).
Cloning and Molecular Biology. Amplification of DNA
segments, from either genomic or plasmid DNA, was accom-
plished using KOD Hot Start DNA polymerase, 8 mM dNTP
stocks (dATP, dTTP, dGTP, and dCTP at 2 mM each), and
KOD Hot Start reaction buffer, all obtained from EMD
Chemicals (Gibbstown, NJ). Polymerase chain reaction (PCR)
conditions were those recommended by the manufacturer, except
for the inclusion of 1% (v/v) dimethyl sulfoxide and 1 M betaine
in the reaction mixtures. The plasmids described in this study (see
Table 1) were stored in and amplified from E. coli strain XL1-
Blue (Stratagene, La Jolla, CA). Qiagen Mini-Prep kits and
QIAquick Spin kits (Qiagen, Valencia, CA) were used to purify
plasmids and DNA fragments, respectively, by protocols de-
scribed by the manufacturer. Restriction endonucleases, T4
DNA ligase, and calf intestinal alkaline phosphatase were
obtained from New England Biolabs (Ipswich, MA). Unless
otherwise stated, the following concentrations of antibiotics were
used when appropriate: 100 μg/mL ampicillin, 50 μg/mL kana-
mycin, 20 μg/mL chloramphenicol, and 15 μg/mL tetracycline.
Construction of Plasmids. DNA oligomers (Integrated
DNA Technologies, Coralville, IA) were used to amplify E. coli
K-12 (NP_414724) lpxH and C. crescentus CB15 (NP_420716)
LpxI from genomic DNA obtained from ATCC (Rockville,
MD). Primers (Table 1 of the Supporting Information) were
designed to confer 5 NdeI and 3 BamHI restriction sites upon the
ends each of the amplified oligonucleotides. A Mastercycler
Gradient Thermocycler (Eppendorf, Hamburg, Germany) was
used to amplify linear DNA. These PCR products were digested
using NdeI and BamHI, according to the manufacturers’ proto-
cols and subsequently ligated into a similarly digested recipient
vector, pET21b (Novage/EMD Chemicals). These ligation reac-
tion mixtures were then transformed into the XL1-Blue strain of
chemically competent E. coli (Stratagene), according to the
manufacturer’s recommended procedure. Plasmids were isolated
from transformants, and their sequences were confirmed by the
Duke Cancer Center DNA Sequencing Facility. The resulting
constructs were designated pEcH21b and pCcH21b (see Table 1).
0
ribosome binding site in the optimal position 7 bp 5 to the kan
cassette’s start codon. The resulting linear oligonucleotide was
electroporated into temperature-shifted E. coli strain DY330
(19), harboring either pBAD30, pBAD30Ec, or pBAD30Cc
[strain DY330VC, DY330Ec, or DY330Cc, respectively (see
Table 1)], as previously described (20). The strains were plated
on LB-agar supplemented with kanamycin and ampicillin and
grown for 18 h at 30 °C. Transformants were repurified, and
colony PCR was performed using primers Kan_FW and
Kan_RV (Table 1 of the Supporting Information) to amplify
the lpxH locus and its 100 bp flanking regions. The resulting
oligonucleotides were sequenced to confirm their identities.
DY330 strains with lpxH::Kan replacements, covered by pBA-
D30Ec and pBAD30Cc, were designated DY330ΔHEc and
DY330ΔHCc, respectively (Table 1).
A P1vir lysate was prepared (21) from DY330ΔHEc and used
to infect E. coli W3110A cells harboring either pBAD33,
pBAD33Ec, or pBAD33Cc (strain W3110AVC, W3110AEc, or
W3110ACc, respectively) (Table 1). Following infection and
outgrowth as previously described (21), cells were spread onto
LB-agar plates supplemented with chloramphenicol, kanamycin,
and 5 mM sodium citrate and allowed to grow for 20 h at 30 °C.
Colonies were selected and repurified twice to remove traces of
contaminating phage. Colony PCR was performed to amplify the
chromosomal region (100 bp from the lpxH locus, and the
resulting oligonucleotides were confirmed by sequencing. The
strains thereby created from the transduction of lpxH::Kan into
W3110ACc and W3110AEc were designated W3110AΔHEc and
W3110AΔHCc, respectively (see Table 1).
0
0
UDP-2,3-diacylglucosamine Hydrolase Expression and
in Vitro TLC Assay. Plasmids pET21b and pCcI21b were
transformed into E. coli strain C41(DE3) by electroporation and
grown for 18 h at 30 °C on LB-agar plates supplemented with

6
718 Biochemistry, Vol. 49, No. 31, 2010
Metzger and Raetz
F
(
IGURE 2: Sequence alignments of LpxH and LpxI orthologues. (A) Sequence alignments of five representative LpxH orthologues.
B) Alignments of five typical LpxI orthologues. In each panel, the absolutely conserved residues are colored red. There is no conservation of
domains or of possible active site motifs between LpxI and LpxH, which are members of distinctly different protein families. The sequences were
obtained from http://www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi.
ampicillin. Single colonies of resulting strains VC_21b and
CcI_21b (see Table 1) were used to inoculate 5 mL overnight
cultures. These, in turn, were used to inoculate 50 mL cultures of
appropriately supplemented LB medium, at an initial OD600 of
cells were grown for an additional 5 h, until the final A600 of the
induced cells was ∼3. Cells were collected by centrifugation at
3000g, washed with phosphate-buffered saline (PBS) (22), and
resuspended in 3 mL of ice-cold PBS. The washed, resuspended
cells were passed twice through a French pressure cell at 18000
psi, and cell debris was removed by centrifugation at 10000g for
30 min. Lysates were analyzed by SDS-PAGE and were assayed
for UDP-2,3-diacylglucosamine hydrolase activity (8).
∼
0.02. Strains VC_21b and CcI_21b were then grown at 30 °C
with aeration at 220 rpm, until the A reached ∼0.5 (∼5 h).
600
Expression was induced by addition isopropyl β-
D-1-thiogalac-
topyranoside (IPTG) to a final concentration of 250 μM. The

Article
Biochemistry, Vol. 49, No. 31, 2010 6719
Table 1: Relevant Strains and Plasmids
strain
description
source
-
-
-
C41(DE3)
W3110A
F
F
ompT hsdS
B
(r
B
m
B
þ
) gal dcm (DE3) D(srl-recA)306::Tn10
51
-
R
aroA::Tn10 msbA , Tet
28
0
R
XL1-Blue
MN7
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F proAB lacIqZDM15 Tn10 (Tet )]
K-12-derived pgsA444 lpxB1; accumulates lipid X
Strategene
52
DY330
W3110 ΔlacU169 gal490 λcI857 Δ(cro-bioA)
19
R
Ec21XB
XL1-Blue harboring pEcH21b, Amp
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
this work
R
Cc21XB
XL1-Blue harboring pCcI21b, Amp
R
EcH33XB
CcI33XB
XL1-Blue harboring pBAD33Ec, Cam
R
XL1-Blue harboring pBAD33Cc, Cam
R
EcH30XB
CcI30XB
XL1-Blue harboring pBAD30Ec, Amp
R
XL1-Blue harboring pBAD30Cc, Amp
R
DY330VC
DY330Ec
DY330Cc
DY330ΔHEc
DY330ΔHCc
W3110AVC
W3110AEc
W3110ACc
W3110AΔHEc
W3110AΔHCc
VC_21b
DY330 harboring pBAD30, Amp
R
DY330 harboring pBAD30Ec, Amp
R
DY330 harboring pBAD30Cc, Amp
R
R
R
DY330 lpxH::Kan harboring pBAD30Ec, Kan , Amp
R
DY330 lpxH::Kan harboring pBAD30Cc, Kan , Amp
R
W3110A harboring pBAD33, Cam
R
W3110A harboring pBAD33Ec, Cam
R
W3110A harboring pBAD33Cc, Cam
R
R
R
W3110A lpxH::Kan harboring pBAD33Ec, Cam , Amp
R
W3110A lpxH::Kan harboring pBAD33Cc, Cam , Amp
R
C41(DE3) harboring pET21b, Amp
R
EcH_21b
CcI_21b
C41(DE3) harboring pEcH21b, Amp
R
C41(DE3) harboring pCcI21b, Amp
plasmid
description
source
R
pET21b
high-copy number expression vector containing a T7 promoter, Amp
Novagen
Novagen
18
R
pET28b
high-copy number expression vector containing a T7 promoter, Kan
R
pBAD30
pBAD33
pEcH21b
pCcI21b
arabinose inducible vector, Amp
R
arabinose inducible vector, Cam
18
R
pET21b containing E. coli lpxH, Amp
this work
this work
this work
this work
this work
this work
R
pET21b containing C. crescentus lpxI, Amp
R
pBAD33Ec
pBAD33Cc
pBAD30Ec
pBAD30Cc
pBAD33 containing E. coli lpxH, Cam
R
pBAD33 containing C. crescentus lpxI, Cam
R
pBAD33 containing E. coli lpxH, Amp
R
pBAD33 containing C. crescentus lpxI, Amp
For our initial analysis, the addition of 5 μL of diluted lysate
from strain VC_21b or CcI_21b was used to start the UDP-2,3-
diacylglucosamine hydrolase reactions in a total volume of 25 μL.
Nonradiolabeled UDP-2,3-diacylglucosamine was prepared as
previously described (8). Briefly, the final reaction mixtures
contained 1 mM UDP-2,3-diacylglucosamine, 20 mM HEPES
water-bath shaker for at least 20 h at 37 °C, with shaking at
100 rpm. The reaction mixture was dried under nitrogen until no
traces of pyridine remained (∼2 h). The residue at the bottom
of the tube was redissolved in 100-300 μL of 20 mM HEPES
(pH 8.0) containing 0.02% (w/v) Triton X-100 and subjected to
bath sonic irradiation for 2 min. This material was split into aliquots
and stored at -80 °C. With this method, we could consistently
(
pH 8.0), and 2 mM MnCl , and they were equilibrated at 30 °C
2
32
32
for 15 min in 0.5 mL polypropylene Eppendorf tubes. Reactions
were started by addition of crude lysates from VC_21b or
CcI_21b, diluted as appropriate with PBS. At various time
points, the reactions were quenched by spotting 5 μL portions
onto 10 cm ꢀ 10 cm high-performance silica TLC plates (Merck,
Darmstadt, Germany). These were developed and dried as
described previously (8). Reaction products were visualized by
spraying the dried TLC plate with 10% H SO in ethanol and
convert more than 95% of [ P]lipid X to [β- P]UDP-2,3-diacyl-
glucosamine.
Expression and Purification of C. crescentus LpxI.
A
colony of E. coli strain CcI_21b (that overexpresses CcLpxI upon
induction) was used to inoculate a 25 mL overnight LB medium
culture supplemented with ampicillin. Following growth for 16 h
at 30 °C, this culture was used to inoculate 1 L of LB broth
containing ampicillin to an initial A
600
of ∼0.05. Growth
2
4
subsequent charring on a hot plate.
proceeded at 30 °C with aeration at 220 rpm, until A reached
600
3
2
32
Preparation of [β- P]UDP-2,3-diacylglucosamine. P-
labeled lipid X was prepared as previously reported (23). Follow-
ing purification, lipid X was redissolved in 2 mL of a CHCl3/
MeOH mixture (2:1, v/v), transferred to a clean screw-capped
glass tube, and thoroughly dried under a nitrogen stream. Next,
∼0.5. IPTG was then added to a final concentration of 250 μM,
and growth was continued for 5 h at 30 °C. Cells, which typically
grew to an A
of ∼3.5, were harvested by centrifugation at
600
3000g for 30 min and subsequently washed with 80 mL of 20 mM
HEPES (pH 8.0) containing 50 mM NaCl. The washed pellets
were stored at -80 °C. In a typical purification, ∼4 g of wet cell
pellet was resuspended in ∼300 mL of lysis buffer [20 mM
HEPES (pH 8.0) containing 50 mM NaCl], and cells were lysed
by three passages through an ice-cold Cell-Cracker pressure
disruption chamber (Microfluidics International Corp., Newton,
10 mg of UMP-morpholidate, 50 μL of 0.2 M 1-H-tetrazole in
acetonitrile, and 1 mL of dry pyridine were added. The tube was
immediately sealed with a Teflon-coated screw cap and subjected
to sonic irradiation for 5 min in a bath sonicator (Avanti Polar
Lipids, Alabaster, AL). The mixture was incubated in a rotary

6
720 Biochemistry, Vol. 49, No. 31, 2010
Metzger and Raetz
MA). Cell debris and membranes were removed by ultracentri-
fugation at 150000g for 1 h. A Rabbit-Plus peristaltic pump
(
Rainin Instruments, LLC, Oakland, CA) was used to load the
membrane-free lysate at ∼2 mL/min onto a 5 mL High-Trap Q
Sepharose Fast-Flow anion exchange cartridge (GE Health
Sciences, Pittsburgh, PA), previously equilibrated in lysis buffer.
The column was washed with 20 volumes (100 mL) of lysis buffer
at 3 mL/min and was then attached to an AKTA 600 FPLC
system (GE Health Sciences). A 30 column volume (150 mL)
continuous gradient from 100% buffer A [20 mM HEPES (pH
8
(
2
.0) containing 50 mM NaCl] to 100% buffer B [20 mM HEPES
pH 8.0) containing 200 mM NaCl] was applied at a flow rate of
mL/min. The protein, which was collected in 5 mL fractions,
began to elute at ∼100 mM NaCl. SDS-PAGE was used to
determine which fractions to pool; typically, six fractions (30 mL)
were combined. The partially purified CcLpxI was concentrated
to ∼10 mL using two 15 mL Amicon Ultra 10000 molecular
weight cutoff centrifuge concentrators (Millipore, Billerica, MA),
which had been washed in buffer B. The AKTA 600 FPLC
system was used to load the concentrated, pooled Q Sepharose
fractions at 1.5 mL/min onto a 330 mL size-exclusion column
[
Superdex 200 XK26/70 (GE Healthcare, Waukesha, WI)], equi-
librated with buffer B. The peak of LpxI eluted at ∼210 mL.
Fractions of 5 mL were collected, and their purity determined by
SDS-PAGE. Typically, four fractions (20 mL) were pooled and
subsequently concentrated to ∼25 mg/mL as described above.
Protein aliquots were stored at -80 °C and subjected to fast
freeze-thaw cycles upon storage and retrieval.
Autoradiographic in Vitro Assay of CcLpxI. Unless
otherwise noted, 25 μL reaction mixtures containing 20 mM
HEPES (pH 8.0), 0.5% (w/v) fatty acid-free bovine serum
albumin (BSA), 0.05% (w/v) Triton X-100, 2 mM MgCl ,
2
32
100 μM UDP-2,3-diacylglucosamine, 1000 cpm/μL [β- P]UDP-
2,3-diacylglucosamine, and enzyme were prepared in 0.5 mL
polypropylene tubes. Prior to addition of 5 μL of enzyme as the
last step, the other reaction components were equilibrated at 30 °C
for 15 min. Unless otherwise noted, enzyme samples were diluted
in a buffer identical to the assay mixture, but lacking UDP-2,3-
diacylglucosamine. At various time points, 3 μL reaction portions
were removed and spotted onto 20 cm ꢀ 20 cm silica gel TLC
plates (EMD Chemicals). These were then developed, dried,
scanned, and quantified as previously described (8).
Kinetic Parameters, pH Optimum, and Detergent De-
pendence of CcLpxI. To determine the effect of pH on the
apparent specific activity of CcLxpI, partially purified enzyme
was assayed as described above, but with the usual 20 mM
HEPES (pH 8.0) replaced with a triple-buffer system consisting
of 100 mM sodium acetate, 50 mM bis(2-hydroxyethyl)iminotris-
F
IGURE 3: Purification of untagged recombinant CcLpxI. (A) Elu-
tionofCcLpxImonitoredatA280 fromasize-exclusioncolumn. (B) A
2% SDS-PAGE analysisofselectedfractions fromthe gel filtration
1
column. The lanes correspond to the elution volume of the trace
shown in panel A. Equal volumes were loaded. (C) SDS-PAGE
analysis of protein from each step of the CcLpxI purification. MFL,
QS, and SZ denote membrane-free lysate, pooled fractions from the
Q-Sepharose anion exchange column, and pooled fractions from the
Superdex S200 sizing column, respectively. Approximately 20 μg of
protein was loaded in each lane.
concentration of Triton X-100 was varied from 0 to 1.0% (w/v).
To measure LpxI activity in the absence of detergent, the
[β- P]UDP-2,3-diacylglucosamine was suspended in buffer lack-
ing Triton X-100. An enzyme concentration of 10 nM was used in
these assays.
(
hydroxymethyl)hexane, and 50 mM Tris (24). CcLpxI activity
32
was measured from pH 4.0 to 9.0. A single-limb pK curve was fit
a
to the data using KaleidaGraph. Typically, the enzyme was
present at a concentration of 5-50 nM.
To determine the KM and Vmax of CcLpxI with respect to
UDP-2,3-diacylglucosamine, the purified enzyme was assayed as
described above, but with the concentration of UDP-2,3-diacyl-
glucosamine varied from 5 to 1000 μM. KaleidaGraph was used
to fit velocities to the Michaelis-Menten equation (25). The
concentration of CcLpxI in the assay was varied from 2 and
Metal Dependence of CcLpxI. To determine whether
divalent metal ions stimulate CcLpxI activity in vitro, the standard
assay condition was employed, except that 2 mM MgCl was
2
replaced by no additive or each of the following chloride salts at
2
þ
2þ
2þ
2þ
2þ
2þ
2.0, 0.2, or 0.02 mM: Mg , Ca , Co , Ni , Cu , Zn , and
2þ
Mn ; a similar EDTA control was also included. The concentra-
tion of CcLpxI in the assay was varied from 2 and 200 nM to follow
linear conversion to product with time in the presence of the
200 nM to maintain linear conversion to product with time at
different UDP-2,3-diacylglucosamine concentrations.
To probe whether CcLpxI activity is affected by detergent,
the typical assay conditions were employed, except that the
different additives. A separate assay, in which MgCl was titrated
from 0 to 20 mM, was performed in the presence of 20 nM CcLpxI.
2

Article
Biochemistry, Vol. 49, No. 31, 2010 6721
Table 2: Purification of CcLpxI from E. coli Strain CcI_21b
protein
(mg)
volume
units
specific activity
yield
(%)
x-fold
(μmol min- mg
1
-1
)
purification
step
(mL)
(mmol/min)
membane-free lysate
168
34
280
30
4.1
0.9
0.8
25
26
30
100
22
1.0
1.1
1.2
Q-Sepharose anion exchange
S200 size exclusion
27
20
20
3
2
F
IGURE 4: Linearity of CcLpxI activity with time and protein concentration. (A) Fraction of 100 μM [β- P]UDP-2,3-diacylglucosamine
converted to the product lipid X at various times under standard assay conditions in the presence of 12.5 nM CcLpxI. (B) Fraction of 100 μM
32
[
β- P]UDP-2,3-diacylglucosamine converted to lipid X per minute as a function of enzyme concentration. (C) Image of a silica TLC plate of an in
vitro CcLpxI reaction run at an enzyme concentration of 40 nM.
Preparation of the CcLpxI Reaction Products in the
1
2 min, followed by a linear gradient from 100% mobile phase A to
8
Presence of H2 O. Reaction mixtures (50 μL each) consisting of
100% mobile phase B consisting of CHCl , methanol, water, and
3
1
00 μM UDP-2,3-diacylglucosamine, 2 mM MgCl , 20 mM
saturated aqueous NH OH (120:68:10:1, v/v/v/v) over 14 min.
2
4
HEPES (pH 8.0), and 66 nM purified CcLpxI were set up in
Next, 100% mobile phase B was applied for 11 min, followed by a
linear gradient from 100% mobile phase B to 100% mobile phase C
16
18
16
the presence of either 100% H2 O or an H2 O/H2 O mixture
77:23, v/v) (Cambridge Isotopes, Andover, MA). The reactions
(
consisting of CHCl , methanol, water, and saturated aqueous
3
were allowed to proceed at 30 °C for 2 h and were quenched by
conversion to a 1.9 mL single-phase, acidic Bligh-Dyer system (26).
A 20 μL portion of this material, containing 37 ng of lipid X, was
analyzed by normal-phase liquid chromatography (LC) and mass
spectrometry. Briefly, the sample was loaded onto a 25 cm ꢀ
NH OH (90:90:19:1, v/v/v/v) over 3 min. Finally, mobile phase C
4
was applied for 3 min, followed by a linear transition to mobile
phase A over 30 s; the latter was passed over the column for an
additional 5 min. The following parameters were used for negative
ion electrospray mass spectrometry (ESI-MS) and MS/MS analysis:
ionization source energy of -4500 V, curtain gas pressure of 20 psi,
GSI of 20 psi, DP of -55 V, and FP of -150 V, with nitrogen used
as the collision gas. The Analyst QS software suite (Applied Bio-
systems) was employed for data analysis.
2
.1 mm Ascentis silica HPLC column (Sigma-Aldrich) using an
Agilent (Santa Clara, CA) 1200 Quaternary LC system, coupled
to a QSTAR XL quadrupole time-of-flight mass spectrometer
(Applied Biosystems, Foster City, CA), operating in the ESI
negative ion mode. Following injection onto the column, which
RESULTS
was equilibrated in mobile phase A consisting of CHCl , methanol,
3
and saturated aqueous NH OH (160:39:1, v/v/v), the sample was
subjected to chromatography at a constant flow rate of 300 μL/min.
First, an isocratic gradient of 100% mobile phase A was applied for
Identification of a Novel UDP-2,3-Diacylglucosamine
Hydrolase in Bacteria Lacking lpxH. The Clusters of Ortho-
logous Genes database (17) was used to compare the genomes of
4

6
722 Biochemistry, Vol. 49, No. 31, 2010
Metzger and Raetz
(
Figure 1). The corresponding gene product, annotated as
DUF1009 in each of these three strains (17), is also present in
many (though not all) of the other organisms that produce lipid A
but lack lpxH. LpxH orthologues display no sequence similarity
to the DUF1009 sequences (Figure 2), which have a distinctly
different set of conserved amino acid residues.
The gene encoding the C. crescentus DUF1009 orthologue,
CC1910 (16), was amplified from genomic DNA, cloned into the
high-copy number pET21b plasmid, and expressed in E. coli
C41(DE3), yielding strain CcI_21b. This construct was grown in
parallel with its empty vector control (VC_21b) and induced to
overexpress the CC1910 protein. Cells from each strain were
harvested and lysed. Overexpression of the expected 29 kDa
CC1910 protein was confirmed by SDS-PAGE (Figure 1A of
the Supporting Information). Equal concentrations of lysates
from VC_21b versus CcI_21b were used to detect UDP-2,3-
diacylglucosamine hydrolase activity by TLC, similar to the
method described for E. coli LpxH (8). This analysis revealed
that the CcI_21b lysate contained >1000-fold higher UDP-2,3-
diacylglucosamine hydrolase activity than its matched vector
control (in Figure 1B of the Supporting Information, lane 2 vs
lane 4), suggesting that CC1910, hereafter termed CcLpxI, is
functionally similar to LpxH. Upon incubation of these lysates
with CDP-diacylglycerol, under otherwise similar assay condi-
tions, no difference in the rate of CDP-diacylglycerol hydrolysis
was observed between extracts of VC_21b and CcI_21b, showing
that LpxI, in contrastto CDP-diacylglucosamine hydrolase(7, 8),
is selective for UDP-2,3-diacylglucosamine (data not shown).
C. crescentus lpxI Can Replace lpxH in E. coli. To
determine whether C. crescentus lpxI possesses UDP-2,3-diacyl-
glucosamine hydrolase activity in vivo, the essential lpxH gene
R
was replaced in E. coli DY330 (19) with a kan cassette, and
covering plasmids expressing either E. coli lpxH or C. crescentus
lpxI were used to test for complementation of lpxH deficiency.
Whereas the empty vector could not complement the deletion of
the chromosomal lpxH gene, plasmids expressing either E. coli
lpxH or C. crescentus lpxI allowed this deletion mutant to form
colonies (Figure 2 of the Supporting Information). The lpxH::
kan deletion could also subsequently be transduced into wild-
type strain E. coli W3110A (28) harboring either E. coli lpxH or
C. crescentus lpxI on a pBAD plasmid. The replacement of lpxH
with kan in W3110A was confirmed by PCR amplification of the
lpxH locus and its 100 bp flanking regions. The identities of these
PCR products were confirmed by sequencing.
F
IGURE 5: Substrate, pH, and detergent dependence of CcLpxI
activity. (A) UDP-2,3-diacylglucosamine concentration dependence
of CcLpxI activity under standard assay conditions. The apparent
Expression and Purification of CcLpxI. Induced cells of
E. coli strain CcI_21b massively overproduced a protein of the
molecular weight expected for LpxI and its associated UDP-2,3-
diacylglucosamine hydrolase activity. To characterize CcLpxI, it
was purified to greater than 90% homogeneity using ion-exchange
and size-exclusion chromatography (Figure 3). The pellet from
K , fit using Kaleidograph, was 105 ( 25 μM; the apparent Vmax was
M
-1 -1
6
9 ( 5 μmol min mg . (B) CcLpxI activity as a function of pH.
The activity is relatively constant between pH 6.5 and 9 but declines
sharply below pH 6. (C) Effect of Triton X-100 concentration on the
apparent specific activity of CcLpxI at 100 μM UDP-2,3-diacylglu-
cosamine. Surface dilution kinetics are not apparent, and there is
measurable activity in the absence of Triton.
5
00 mL of CcI_21b cells grown to an A600 of ∼3.5 yielded ∼30 mg
of CcLpxI. The specific activity of the protein increased only 1.2-
fold during the purification, consistent with the efficient over-
expression, and the total activity yield was ∼20% (Table 2).
CcLpxI elutes from a gel filtration column in a symmetric peak
(Figure 3A), consistent with either a dimeric or monomeric solu-
tion structure. Unlike EcLpxH, which is a peripheral membrane
protein, recombinant CcLpxI did not sediment with membranes
but remained in the supernatant following ultracentrifugation.
Gram-negative bacteria lacking lpxH orthologues. Given that the
gene(s) encoding a novel UDP-2,3-diacylglucosamine hydrolase
analogue(s) might be located near other lipid A biosynthetic
genes and that in many species lpxD, fabZ, lpxA, and lpxB cluster
in an operon, we searched for species that lacked lpxH but
possessed additional open reading frames of unknown function
in the lpxD-fabZ-lpxA-lpxB locus. Several bacteria, including
C. crescentus (16), Sinorhizobium meliloti (27), and M. loti (15),
met this criterion in that an ∼1000 bp open reading frame
of unknown function was inserted between lpxA and lpxB
3
2
An Autoradiographic Assay of CcLpxI Using [β- P]UDP-
32
2,3-Diacylglucosamine. Using [β- P]UDP-2,3-diacylglucosa-
mine as the substrate, we developed a quantitative TLC-based

Article
Biochemistry, Vol. 49, No. 31, 2010 6723
F
IGURE 6: Metal dependence of CcLpxI. (A) Specific activity of CcLpxI in the presence of various divalent cations or EDTA. Three sets of assays
were performed, each of which included a no additive (No add) control, as shown at the left. Each bar in the series of three bars represents a
different concentration of each additive (e.g., 0.02, 0.2, or 2 mM). Standard assay conditions were used with an enzyme concentration of 10 nM.
(
2
B) The standard CcLpxI assay was used with 10 nM enzyme, but with the MgCl concentration increasing from 0 to 20 mM.
2
þ
2þ
assay for CcLpxI. Enzymatic activity is linear with time and with
protein concentration (Figure 4A,B). Moreover, CcLpxI quanti-
tatively converts [β- P]UDP-2,3-diacylglucosamine to [ P]lipid
X (Figure 4C).
into reaction mixtures containing 2 mM Mg or Mn (data not
shown). Inhibition was also seen at the higher concentrations of
3
2
32
2þ
2þ
.
Cu and Zn
Next, LpxI activity was measured under standard assay condi-
tions, but with the concentration of Mg varied from 0 to 20 mM.
2þ
Apparent Kinetic Parameters and Detergent Depen-
dence of CcLpxI. The apparent KM of purified CcLpxI with
respect to UDP-2,3-diacylglucosamine was 105 ( 25 μM, while
2þ
CcLpxI activity was maximal at 0.2 mM Mg and remained
2
þ
approximately the same up to 20 mM Mg (Figure 6B). To
determine whether divalent cations are associated with puri-
fied CcLpxI, inductively coupled plasma mass spectrometry
was performed. No significant amounts of stoichiometric,
tightly bound metal ions were detected in the purified protein
in two separate determinations.
the apparent Vmax was 69 ( 5 μmol min- mg (Figure 5A). The
pH-rate profile was determined for CcLpxI, and a single pKa
curve was fit to the data using Kaleidograph. The pKa was
estimated to be 6.15 ( 0.86 (Figure 5B), although the data deviate
from a single-ionization model at low pH. The detergent depen-
dence of CcLpxI was determined by varying the concentration of
Triton X-100 in the assay from 0 to 1% (w/v). While stimulated
1
-1
CcLpxI Catalyzes the Attack of H O on the β-Phos-
2
phorus Atom of UDP-2,3-Diacylglucosamine. To determine
which phosphorus atom of UDP-2,3-diacylglucosamine is
attacked during hydrolysis catalyzed by CcLpxI, excess purified
enzyme was used to convert UDP-2,3-diacylglucosamine to pro-
∼
3-fold in the presence of 0.05% (w/v) Triton X-100, the appa-
rent activity did not decrease at high concentrations of Triton
X-100 (Figure 5C), showing that CCLpxI activity is not appa-
rently decreased by surface dilution of the substrate (29).
Metal Dependence of CcLpxI Activity in Vitro. The
purified CcLpxI was assayed under the standard conditions,
except that each of the following chloride salts was added at
1
6
18
16
ducts quantitatively in the presence of H2 O or an H2 O/H2
O
mixture (77:23, v/v). Lipid X and UMP products were separated
by normal-phase LC and analyzed by ESI-MS in the negative ion
mode (Figure 7A,B). Lipid X (predicted [M - H]- at m/z
710.424) eluted between minutes 24.4 and 24.6; the UMP
emerged during column regeneration between minutes 38.3 and
2þ 2þ 2þ 2þ 2þ 2þ
0.02, 0.2, or 2 mM: Mg , Ca , Co , Ni , Cu , Zn , and
2þ
Mn (explained in the legend of Figure 6). For each concentra-
tion, a control assay was included in which divalent cations were
not added (Figure 6A, no add); in another set, 0.02, 0.2, or 2 mM
EDTA was included in place of the divalent cations. The
apparent specific activity of purified CcLpxI increased 10-fold
1
8
39.0. For the H2 O-labeled reaction, the proportional amount
1
8
(∼77%) of the lipid X product contained O, as shown by the
presence of a much more intense peak at m/z 712.441 versus m/z
-
710.440 (Figure 7B). This result is expected for the [M - H] ion
2þ
18
in the presence of Mg versus no added metal, or 6-fold in the
of a lipid X molecule containing a single O atom (predicted
2þ
2þ
-
16
presence of added Mn or Co (Figure 6A). The presence of
EDTA at either 2.0 or 0.2 mM completely inhibited CcLpxI. This
inhibition was reversed by dilution of the EDTA-treated enzyme
[M - H] at m/z 712.429) versus lipid X containing only
O
-
18
(predicted [M - H] at m/z 710.424). No O was incorpo-
1
8
rated into the UMP product isolated from the CcLpxI
O

6
724 Biochemistry, Vol. 49, No. 31, 2010
Metzger and Raetz
protein (Figure 1A of the Supporting Information). E. coli lysates
of strain CcI_21b displayed >1000-fold increased UDP-2,3-
diacylglucosamine hydrolase activity relative to vector controls
(
Figure 1B of the Supporting Information). The presence of
CC1910, renamed lpxI, permitted the deletion and replacement
of the chromosomal lpxH gene with a kanamycin resistance
cassette in E. coli (Figure 2 of the Supporting Information). These
results demonstrate that LpxI functions as an alternative UDP-
2
,3-diacylglucosamine hydrolase in vivo.
Taken together with the dissimilarity of their conserved motifs
(Figure 2), we propose that EcLpxH and CcLpxI evolved sepa-
rately to generate the same products by different hydrolytic
mechanisms. The presence of lpxI, but not lpxH, in more ancient
species like A. aeolicus, suggests that lpxI may have evolved earlier
than lpxH. Moreover, some groups of organisms that make lipid
A, such as the Cyanobacteria (33) and the Chlamydiae (34, 35),
lack both lpxH and lpxI, suggesting that at least one additional
class of UDP-2,3-diacylglucosamine hydrolases may exist.
To determine which P atom of UDP-2,3-diacylglucosamine is
attacked by water during CcLpxI-catalyzed hydrolysis, reactions
1
8
were run in the presence or absence of H2 O. Analysis of the
18
products by ESI-MS (Figure 7) revealed that the O was incorpo-
rated exclusively into lipid X. CcLpxI therefore catalyzes the attack
of water on the β-P atom, whereas EcLpxH specifically attacks the
R-P atom (Scheme 2) (8). This observation, together with the lack of
homology between EcLpxH and CcLpxI, strongly suggests that
these enzymes perform catalysis by different mechanisms.
18
The incorporation of H2 O into the β-P atom of UDP-2,3-
diacylglucosamine by LpxI is formally analogous to the chem-
istry performed by phosphatidylserine synthase (PssA) of E. coli,
which catalyzes the transfer of the phosphatidyl moiety of CDP-
diacylglycerol to the hydroxyl group of serine (36). PssA catalysis
is thought to proceed via a phosphatidyl-enzyme intermedi-
ate (37), but PssA can also cleave CDP-diacylglycerol to generate
phosphatidic acid and CMP in the absence of serine, with
1
8
F
lyzed hydrolysis of UDP-2,3-diacylglucosamine in 77% H
IGURE 7: Incorporation of O into lipid X during CcLpxI-cata-
18
2
O.
CcLpxI-catalyzed hydrolysis of UDP-2,3-diacylglucosamine was
conducted in the absence or presence of 77% H O. (A) LC-ESI-
MS analysis of lipid X generated by CCLpxI in the presence of 100%
18
concomitant incorporation of H2 O into the phosphatidic acid
18
2
product by attack of water on the β-P atom of CDP-diacylgly-
cerol (36). This consideration raises the intriguing converse
possibility that LpxI (or other enzymes like it) might be able to
transfer the 2,3-diacylglucosamine 1-phosphate moiety from
UDP-2,3-diacylglucosamine to an acceptor molecule other than
water, analogous to the process of phosphatidyl transfer in
glycerophospholipid metabolism.
1
2
6
H
O. (B) LC-ESI-MS analysis of lipid X generated by CCLpxI in
18 16
the presence of an H
2 2
O/H O mixture (77:23, v/v). The insets show
LC-ESI-MS analyses of the UMP generated in the reactions.
reaction mixture (Figure 7B, inset). We conclude that ccLpxI
catalyzes the attack of water exclusively on the β-phosphorus
atom of UDP-2,3-diacylglucosamine (Scheme 2).
The significance of two mechanisms for cleaving UDP-2,3-
diacylglucosamine remains unclear. Phosphohydrolases are ubi-
quitous in biology and have probably evolved many times on
different protein scaffolds (38). Phosphohydrolases are often metal-
dependent, and much is known about their mechanisms (39). They
play diverse roles in the modification and processing of pro-
teins (40, 41), nucleic acids (42, 43), lipids (44-47), and diverse
small molecules (39, 48-50). The mechanisms of these enzymes
generally involve the positioning of a deprotonated water molecule
for attack on a P atom of a coordinated phosphate moiety of the
substrate (39). The roles played by the metal ions may vary. In
some cases, the metal coordinates the oxygen atoms of the
phosphate group, while in others, it positions the water for attack
on the P atom (39, 48-50). In still other instances, the metal
appears to coordinate a network of ordered water molecules, which
in turn position a “catalytic water” for attack on the substrate (48).
Although purified CcLpxI does not contain any tightly bound
divalent cations, it is dependent upon them for activity (Figure 6A).
Structural and mechanistic studies will be required to elucidate the
DISCUSSION
Although essential for growth in E. coli and present in most
γ- and β-proteobacteria (8, 30), LpxH is missing in R-proteobacteria
and many other groups (Figure 8 and Table 1 of the Supporting
Information). Here, we describe the identification of a different
UDP-2,3-diacylglucosamine hydrolase present in many of the
species that lack LpxH, including almost all R-proteobacteria,
most strains of Leptospira (31), and many diverse organisms, like
A. aeolicus (32). The relevant gene, which encodes a protein of
unknown function annotated as DUF1009, was recognized
because of its location between lpxA and lpxB in C. crescentus
and M. loti (Figure 1) (15, 16). Members of the DUF1009 family
possess no sequence similarity to EcLpxH or any other type of
protein (Figure 2A,B).
We cloned the gene encoding DUF1009 from C. cresentus
(CC1910) into a high-copy number vector and expressed it in
E. coli, resulting in massive overproduction of the expected 29 kDa

Article
Biochemistry, Vol. 49, No. 31, 2010 6725
F
IGURE 8: Distribution of LpxH and LpxI orthologues among diverse bacterial groups. This figure, modified from the NCBI bacterial genomes
website (http://www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi), shows the approximate distribution of putative LpxH (cyan) and LpxI orthologues
yellow) in diverse bacterial groups for which complete genome sequences are currently available. Light green shading indicates approximately
(
equal proportions of organisms having putative LpxH and LpxI orthologues. Important Gram-negatives, such as the Chlamydiae and the
Cyanobacteria, contain neither protein, even though both possess LpxC (Table 2 of the Supporting Information) and LpxB (Scheme 1). Many
Spirochaetales, such as Treponema pallidum and Borrelia burgdorferi, do not synthesize lipid A. However, species of Leptospira do make lipid A
and contain LpxI orthologues.
roles of metals in CcLpxI catalysis and the basis for the selectivity of
CcLpxI for UDP-2,3-diacylglucosamine.
6. McClerren, A. L., Endsley, S., Bowman, J. L., Andersen, N. H.,
Guan, Z., Rudolph, J., and Raetz, C. R. H. (2005) A slow, tight-
binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A
biosynthesis with antibiotic activity comparable to ciprofloxacin.
Biochemistry 44, 16574–16583.
ACKNOWLEDGMENT
7
. Babinski, K. J., Kanjilal, S. J., and Raetz, C. R. H. (2002) Accumula-
tion of the lipid A precursor UDP-2,3-diacylglucosamine in an
Escherichia coli mutant lacking the lpxH gene. J. Biol. Chem. 277,
We thank Drs. Hak Suk Chung, Jinshi Zhao, and Ziqiang Guan
for their careful reading of the manuscript and other members of
our laboratory for their helpful discussion and criticism. The metal
analysis of LpxI was conducted by Dr. Aaron Atkinson of the Divi-
sion of Hematology at the University of Utah, Salt Lake City, UT.
25947–25956.
8
. Babinski, K. J., Ribeiro, A. A., and Raetz, C. R. H. (2002) The
Escherichia coli gene encoding the UDP-2,3-diacylglucosamine pyropho-
sphatase of lipid A biosynthesis. J. Biol. Chem. 277, 25937–25946.
. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang,
Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and
PSI-BLAST: A new generation of protein database search programs.
Nucleic Acids Res. 25, 3389–3402.
9
SUPPORTING INFORMATION AVAILABLE
Primers used to construct various LpxI overexpressing strains
1
1
0. Price, N. P. J., Kelly, T. M., Raetz, C. R. H., and Carlson, R. W.
1994) Biosynthesis of a structurally novel lipid A in Rhizobium
leguminosarum: Identification and characterization of six metabolic
steps leading from UDP-GlcNAc to (Kdo) -lipid IV . J. Bacteriol.
76, 4646–4655.
1. Gonzalez, V., Santamaria, R. I., Bustos, P., Hernandez-Gonzalez, I.,
Medrano-Soto, A., Moreno-Hagelsieb, G., Janga, S. C., Ramirez,
M. A., Jimenez-Jacinto, V., Collado-Vides, J., and Davila, G. (2006)
The partitioned Rhizobium etli genome: Genetic and metabolic re-
dundancy in seven interacting replicons. Proc. Natl. Acad. Sci. U.S.A.
(
Table 1), additional information regarding the distributions of
(
LpxI and LpxI among diverse bacteria (Table 2), additional
information about the purification of LpxI (Figure 1), and
additional information about the ability to lpxI to complement
the E. coli lpxH mutation (Figure 2). This material is available
free of charge via the Internet at http://pubs.acs.org.
2
A
1
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