THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 34, pp.
24489 –24495, August 25, 2006
Structural Studies of FlaA1 from Helicobacter pylori Reveal
the Mechanism for Inverting 4,6-Dehydratase Activity*□
Received for publication, March 14, 2006, and in revised form, April 27, 2006 Published, JBC Papers in Press, May 1, 2006, DOI 10.1074/jbc.M602393200
Noboru Ishiyama‡1, Carole Creuzenet§2, Wayne L. Miller¶3, Melinda Demendi§, Erin M. Anderson¶, George Harauz¶,
Joseph S. Lam¶4, and Albert M. Berghuis‡储5
From the ‡Department of Biochemistry and 储Department of Microbiology and Immunology, McGill University, Montreal, Quebec
H3A 2B4, the §Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1,
and the ¶Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
FlaA1 from the human pathogen Helicobacter pylori is an
enzyme involved in saccharide biosynthesis that has been shown
to be essential for pathogenicity. Here we present five crystal
structures of FlaA1 in the presence of substrate, inhibitors, and
bound cofactor, with resolutions ranging from 2.8 to 1.9 Å.
These structures reveal that the enzyme is a novel member of the
short-chain dehydrogenase/reductase superfamily. Additional
electron microscopy studies show the enzyme to possess a hex-
americ doughnut-shaped quaternary structure. NMR analyses
of “real time” enzyme-substrate reactions indicate that FlaA1 is
a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the
enzyme catalyzes the first step in the biosynthetic pathway of a
pseudaminic acid derivative, which is implicated in protein gly-
cosylation. Guided by evidence from site-directed mutagenesis
and computational simulations, a three-step reaction mecha-
nism is proposed that involves Lys-133 functioning as both a
catalytic acid and base.
Helicobacter pylori is a spiral shaped, motile, microaerophilic
Gram-negative bacterium that resides in the gastric mucus
layer or adheres to the epithelial lining of the stomach (1). It has
been estimated that half to two-thirds of the world’s population
is chronically infected with H. pylori, and prevalence among
* This work was supported in part by Collaborative Health Research Projects
Grant 251007-02 from the Natural Sciences and Engineering Research
Council of Canada (to J. S. L., C. C., and A. M. B.) and a Natural Sciences and
Engineering Research Council of Canada operating grant (to G. H.). The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code ) have been deposited in the
Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rut-
gers University, New Brunswick, NJ (http://www.rcsb.org/). 2GN4, 2GN6,
2GN8, 2GN9, and 2GNA.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table 1 and supplemental Figs. 1 and 2.
1
Recipient of a studentship from the Canadian Cystic Fibrosis Foundation.
2
Recipient of a University Faculty Award from the Natural Sciences and Engi-
neering Research Council of Canada and a Premier’s Research Excellence
Award from the Province of Ontario.
3
Recipient of a Doctoral Research Award from the Canadian Institute of
Health Research.
4
Holds Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology.
5
Holds Canada Research Chairs in Structural Biology. To whom correspond-
ence should be addressed: Depts. of Biochemistry and Microbiology and
Immunology, McGill University, 740 Dr. Penfield Ave., Rm. 5202, Montreal,
Quebec H3A 1A4, Canada. Tel.: 514-398-8795; Fax: 514-398-2036; E-mail:
albert.berghuis@mcgill.ca.
AUGUST 25, 2006 • VOLUME 281 • NUMBER 34
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
S
adults in developing countries is typically 80 –90% (2). The bac-
terium can asymptomatically remain in the human stomach for
decades; however, infections can lead to gastric inflammation
and ulceration. In addition to its well established etiological role
in several gastroduodenal diseases, H. pylori infection has also
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been implicated in the development of gastric cancer (3, 4). The
significance of the discovery that H. pylori plays a causal role in
gastritis and ulceration was recently recognized through the
awarding of the 2005 Nobel Prize in Physiology or Medicine to
Drs. Marshall and Warren.
The pathogenicity of H. pylori can be attributed to numerous
virulence factors that allow for host colonization, among these are
urease, adhesins, flagella, and lipopolysaccharides (LPS)6 (5–7).
For example, the flagella enable H. pylori to penetrate the gastric
mucus layer and reach the epithelial cells, and LPS O antigens
employ mimicry to Lewis blood group antigens to facilitate subse-
quent cell adhesion and colonization (8). Because of drug resist-
ance and an increase in treatment failure, the biochemical path-
ways responsible for the synthesis and delivery of these virulence
factors have received much attention as they contain potential tar-
gets for drug development (9, 10).
The flaA1 (HP0840) gene product has been shown to be
involved in the synthesis of both flagella and LPS, and as such it
plays a critical role in H. pylori pathogenesis and colonization.
Specifically, disruption of the flaA1 gene results in bacteria
devoid of flagella and with altered LPS that lacks most of the O
antigen (9, 10). FlaA1 is a 37-kDa protein whose sequence sug-
gests that it is a member of the short-chain dehydrogenase/
reductase (SDR) superfamily. Several biochemical studies have
shown that the substrate of FlaA1 is UDP-linked N-acetylglu-
cosamine (UDP-GlcNAc; UDP-2-acetamido-2-deoxy-␣-D-glu-
cose). However, these same studies present conflicting data on
the reaction product produced by FlaA1, hence characterizing
the enzyme as a 4,6-dehydratase (11), a 4,6-dehydratase-4-
reductase (10, 11), or a 4,6-dehydratase-5-epimerase (12).
Unfortunately, the sequence of FlaA1, even given the structural
conservation within the SDR superfamily, provides no indica-
tions on the nature of the catalyzed reaction. In fact, it is unclear
for any of the three suggested catalytic functions what a possi-
ble reaction mechanism could be. The resulting confusion
impacts the identification of the pathway in which the enzyme
6
The abbreviations used are: LPS, lipopolysaccharide; SDR, short-chain
dehydrogenase/reductase; MES, 2-morpholineethanesulfonic acid; Pse,
pseudaminic acid.
JOURNAL OF BIOLOGICAL CHEMISTRY 24489
Structure and Mechanism of Inverting 4,6-Dehydratase FlaA1
TABLE 1
Data collection and refinement statistics
Coenzyme NADPH NADPⴙ NADPⴙ NADPⴙ NADPⴙ
Substrate UDP-GlcNAc UDP-GlcNAc UDP UDP-Glc UDP-Gal
Space group P63 P63 P63 P63 P63
Cell dimensions a (⫽b), c (Å) 111.4, 108.0 109.8, 108.0 111.1, 107.8 112.4, 107.3 112.2, 107.4
Wavelength (Å) 1.100 0.9803 1.100 0.9795 1.100
Resolution (Å) 50-1.9 50-2.7 50-2.1 50-2.8 50-2.6
Completeness (%)a 95.7 (80.5) 97.6 (93.3) 97.9 (93.2) 96.8 (84.6) 97.5 (92.0)
Rsym(I ) (%)a,b 4.9 (42.2) 9.5 (29.5) 6.9 (40.9) 8.2 (26.4) 8.3 (40.5)
I/␴Ia 16.0 (2.0) 8.8 (2.8) 11.3 (1.9) 8.7 (3.3) 10.0 (2.2)
Redundancya 7.4 (4.8) 4.4 (4.3) 8.5 (7.5) 6.5 (5.6) 6.4 (6.4)
No. of reflections 57,284 19,888 43,145 18,312 23,091
Rwork /Rfree (%)c 19.7/22.9 20.4/26.9 20.9/24.5 22.0/30.7 19.6/25.0
No. of atoms/mean B-factor (Å2) 5669/39.4 5489/32.8 5609/40.2 5397/50.6 5516/44.6
Protein 5163/39.7 5163/32.9 5163/40.5 5163/50.7 5163/44.8
Ligand 198/36.7 158/45.1 158/42.4 158/58.7 180/55.5
Solvent 308/36.7 168/18.7 288/33.6 76/29.2 173/29.9
r.m.s.d.d bond lengths (Å) 0.006 0.007 0.006 0.007 0.007
r.m.s.d. Bond angles (°) 1.4 1.5 1.4 1.5 1.4
a
The values in parentheses are for highest resolution shells.
b
Rsym ⫽ ⌺兩I ⫺ 具I典兩/⌺I, where 具I 典 is the mean intensity of all symmetry-related reflections I.
c
Rcryst ⫽ ⌺兩Fo ⫺ Fc兩/⌺Fo. Rfree, as for Rcryst using a random subset of the data (10%) not included in the refinement.
d
r.m.s.d. is root mean square deviation.

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participates and how this pathway plays a role in virulence fac-
tor biosynthesis. Furthermore, it hampers efforts to exploit
FlaA1 as a potential antibiotic target. To address these ques-
tions, we have performed structural biological studies of FlaA1,
using a combination of x-ray diffraction, electron microscopy,
site-directed mutagenesis, and NMR spectroscopy as well as
computational simulations and bioinformatics analyses.
EXPERIMENTAL PROCEDURES
Crystallography—FlaA1 was purified as described previously
(11). A portion of the purified sample was subjected to chemical
reduction to prepare the FlaA1䡠NADPH sample based on a
method described by Thoden et al. (13). Crystals of five differ-
ent ternary complexes of FlaA1 were grown at room tempera-
ture by the hanging-drop vapor diffusion, using as reservoir
solution 10% (v/v) polyethylene glycol-200, 100 mM MES, pH
6.0, 5% (v/w) polyethylene glycol-3000, and 4% acetone, and
providing 10 m excess of substrate. For the crystallization of
the FlaA1䡠NADP⫹䡠UDP-GlcNAc and FlaA1䡠NADP⫹䡠UDP-
Glc ternary complexes, 5 M excess of NAD⫹ was also in-
cluded in the crystallization condition. Note that crystals of
the FlaA1䡠NADPH䡠UDP-GlcNAc ternary complex had a dis-
tinct yellow color reflecting the oxidation state of the cofactor.
Diffraction data were collected at the X8C beam line of the
National Synchrotron Light Source, Brookhaven National Lab-
oratories, and processed using the HKL suite of programs (14).
Statistics pertaining to the diffraction data are presented in
Table 1.
The structure of FlaA1䡠NADP⫹䡠UDP-GlcNAc was deter-
mined by molecular replacement with CNS (15) using the
N-terminal region of WbpP from Pseudomonas aeruginosa
(PDB 1SB8; 16) as a search model. Exploiting the presence of
2-fold noncrystallographic symmetry, the remaining C-termi-
nal region of FlaA1 was built over successive rounds of refine-
ment by mask-less averaging of ␴A-weighted difference elec-
tron density maps with RAVE (17). Crystal structures for FlaA1
in complex with other sugar nucleotides were subsequently
determined by difference Fourier methods. Final refinement
statistics are presented in Table 1.
Electron Microscopy—Purified FlaA1 was adsorbed to a car-
bon-coated Formvar film on a 400-mesh copper electron
microscope grid, washed with double-distilled water, nega-
tively stained with NANO-WTM (Nanoprobes, Yaphank, NY),
blotted, and allowed to air-dry. Electron micrographs were
taken on a Philips CM10 transmission electron microscope
under low dose conditions at a nominal magnification of
⫻73,000 and recorded on Kodak Electron Microscope Film
4489. Micrographs were subsequently digitized at 4800 ⫻ 2400
dpi and analyzed using IMAGIC-V electron image processing
software (18). Briefly, 592 smaller images comprising a single,
defined FlaA1 complex were selected from micrographs and
subjected to standard “single particle analysis” to define repro-
ducible projection views (19).
Site-directed Mutagenesis and Activity Assay—Construction
of mutants, protein production, and activity assays for FlaA1
K133E and K133M variants followed procedures described pre-
viously (20). Circular dichroism spectroscopy, employing a
Jasco J-600 spectropolarimeter, was used to assess that muta-
tions did not affect protein folding. For activity assays the pro-
duction of product was measured by capillary electrophoresis
analysis as described previously (11).
Nuclear Magnetic Resonance Spectroscopy—As the product
of FlaA1 may be labile, the FlaA1 reaction was monitored with
NMR, and the reaction product was identified directly in aque-
ous reaction buffer without purification. FlaA1 protein (90 ␮g)
was suspended in 200 ␮l of 90% H2O, 10% D2O sodium phos-
phate buffer (25 mM NaPO4, 100 mM NaCl, pH 7.2) and placed
in a 3-m[sacp]m NMR tube. To begin the reaction, 5 mM UDP-
GlcNAc was added. The enzymatic reaction was monitored
using 1H NMR analysis (25 °C) at 500 MHz (1H) with a Varian
Inova spectrometer (Varian, Palo Alto) equipped with a Varian
Z-gradient 3-m[sacp]m triple resonance (1H, 13C, 31P) probe.
The reaction was stopped after 3 h when the concentrations of
starting material and product were equal by removing the
FlaA1 protein. The structure of the product was then examined
using standard homo- and heteronuclear correlated NMR
pulse sequences (from Varian) and selective one-dimensional

24490 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 34 • AUGUST 25, 2006
 Structure and Mechanism of Inverting 4,6-Dehydratase FlaA1

FIGURE 1. Structures of the protomer and hexamer of FlaA1. A, ribbon representation of the FlaA1 protomer complexed with NADPH (gray) and UDP-GlcNAc
(black). The ribbon is colored blue to red, corresponding to the N and C termini, respectively. B, ribbon representation of the FlaA1 hexamer with protomers
shown in different colors. C, the class-averaged representative electron microscopy image of the doughnut-shaped FlaA1 protein particles. D, refined image of
C after 3-fold symmetry averaging. E, the contoured image of D. F, superposition of the hexameric FlaA1 crystal structure over E, using the identical scale for

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both objects. All scale bars correspond to 2 nM. A and B and similar representations were prepared using the PyMOL Molecular Graphics System (DeLano
Scientific, San Carlos, CA).

total correlation spectroscopy and nuclear Overhauser effect
spectroscopy experiments (12).
Computational Analyses—Values of the pKa for three active
site residues (Asp-132, Lys-133, and Tyr-141) were computed
using WHATIF (21–23), where DelPhi was employed to calcu-
late the linearized Poisson-Boltzmann equation (24). For
ligands, partial charges were assigned with SYBYL 6.9 (Tripos
Inc., St. Louis) using the MMFF94 (25) force field, and topology
files were generated with PRODRG (26). In the calculations, the
dielectric constants for protein and solvent were set to 8 and 80,
respectively, and the ionic strength of the solvent was placed at
0.144 mM. Besides the three residues listed above, an additional
21 of 94 titratable residues were included in full pKa calcula-
tions (22, 27).
To examine pKa changes during catalysis, calculations were
performed on five models, representing the distinct states along
the reaction coordinate: holoenzyme, substrate-bound state,
first reaction intermediate following oxidation, second reaction
intermediate following water elimination, and product-bound
state.
RESULTS
Overall Structure and Oligomeric Arrangement of FlaA1—
The three-dimensional structure of FlaA1 is bilobal in shape
(Fig. 1A). The larger, predominantly N-terminal, lobe consists
of residues 1–174, 208 –234, and 265–317 and contains a
10-stranded mostly parallel ␤-sheet flanked on either side by
eight helices. The smaller, predominantly C-terminal, lobe is
composed of residues 175–207, 235–264, and 318 –333 and
possesses three ␣-helices and two short two-stranded ␤-sheets.
There are a total of five crossover points between the two lobes,
resulting in an intertwined connectivity. The fold of FlaA1 con-
firms that this enzyme is a member of the SDR superfamily.
However, FlaA1 is unique in that within the larger lobe the
six-stranded parallel ␤-sheet of the Rossmann fold has been
extended by four additional strands.
Biophysical studies for FlaA1 have suggested that under
physiological conditions the enzyme is organized in a higher
order structure; gel filtration experiments suggest that the
enzyme is a dimer (11), whereas dynamic light scattering stud-
ies of purified samples predict at least a tetrameric arrangement
(data not shown). Analysis of crystal packing interactions also
suggests that FlaA1 does not exist as a monomer under physi-
ological conditions. The FlaA1 protomer possesses two
extended buried interfaces within the crystal lattice, both
located on the larger lobe of the molecule, positioned as a
wedge. The first interface buries ⬃3300 Å2 of surface area and is
predominantly hydrophobic in nature. The second interface
buries ⬃2700 Å2 of surface area and incorporates 4 – 6 salt
bridges. Because of the combination of crystallographic and
noncrystallographic symmetry, six FlaA1 protomers form a
compact doughnut in which ⬃20% of the total surface area is
buried (Fig. 1B). To examine the physiological relevance of this
hexameric arrangement, electron microscopy was performed
using a diluted FlaA1 sample. Doughnut-shaped protein parti-
cles were observed in negatively stained micrographs. Subse-
quent single-particle analysis revealed FlaA1 oligomers that are
in excellent agreement with the hexameric structure derived
from crystallographic analysis, confirming that the enzyme
exists as a hexamer in solution (Fig. 1, C–F ).
Active Site Architecture and Catalytic Residues—The FlaA1
crystal structures reveal the cofactor and substrate-binding
sites to be located in the larger and smaller lobes, respectively.
The cofactor for FlaA1 is identified as NADP⫹/NADPH, which
binds in a canonical manner to the Rossmann fold portion of
the predominantly N-terminal lobe. Most intriguingly, exoge-
nous NADP⫹ was not included as part of the crystallization
conditions, indicating that NADP⫹ was co-purified and thus
must bind tightly to the enzyme. In fact, supplementing the
crystallization conditions with excess NAD⫹ did not dislodge
the NADP⫹. This is in agreement with data from biophysical
studies where addition of exogenous cofactor had no effect on
the catalytic activity of FlaA1 (11).
Crystal structures of FlaA1 were obtained with the substrate
UDP-GlcNAc (with reduced and oxidized cofactor) and with

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Structure and Mechanism of Inverting 4,6-Dehydratase FlaA1

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FIGURE 2. Active site architecture. A, the active site structure (green) in the
FlaA1䡠NADPH䡠UDP-GlcNAc abortive ternary complex contains UDP-GlcNAc in
a catalytically competent orientation. The Fo ⫺ Fc SA-omit map (blue mesh) of
UDP-GlcNAc is contoured at 2.0␴. B, the active site structure (orange) in the
FlaA1䡠NADP⫹䡠UDP-Gal ternary complex contains UDP-Gal in an inhibitory ori-
entation. The Fo ⫺ Fc SA-omit map (purple mesh) of UDP-Gal is contoured at
2.0␴. NADPH and NADP⫹ are shown in gray.

UDP, UDP-glucose (UDP-Glc), and UDP-galactose (UDP-Gal).

In all five structures well defined density was observed for the
UDP moiety whose uracil group forms an H-bond to the car-
bonyl oxygen of Pro-197 and whose diphosphate group is sta-
bilized by two arginine residues (205 and 258) and a helix
dipole moment. However, density for the saccharide moiety
was only seen in the FlaA1䡠NADPH䡠UDP-GlcNAc and
FaA1䡠NADP⫹䡠UDP-Gal ternary complexes (Fig. 2). It is not
uncommon that saccharide density is lacking or ill defined in
SDR enzymes that bind UDP-hexoses as this moiety must be
inherently mobile for catalysis to proceed (16, 28, 29). However,
the FlaA1䡠NADPH䡠UDP-GlcNAc structure provides insight on
the average location of the hexose moiety of the substrate in
FlaA1, which is in close proximity to the nicotinamide group of
the cofactor, with both rings being parallel to each other. Sur-
rounding the GlcNAc moiety are several residues that most FIGURE 3. Reaction mechanism for FlaA1 inverting 4,6-dehydratase activ-
likely play a role in catalysis, specifically Thr-131 and Tyr-141 of ity. The proposed reaction mechanism for FlaA1 consists of three sequential
the (S/T)YK triad commonly found in SDR enzymes, and Asp- steps as follows: oxidation, dehydration, and reduction. Computed pKa values for
the active site residues Asp-132, Lys-133, and Tyr-141 are tabulated for each of
132 and Lys-133 (Fig. 2A). Previously, the importance of Tyr- these steps, and the appropriate protonation states are reflected in the
141 for catalysis was illustrated through site-directed mutagen- mechanism.

24492 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 34 • AUGUST 25, 2006
 Structure and Mechanism of Inverting 4,6-Dehydratase FlaA1
FIGURE 4. Proposed pathway for the biosynthesis of CMP-Pse5Ac7Ac in H. pylori. Six biosynthetic enzymes are involved in the production of CMP-
Pse5Ac7Ac from UDP-GlcNAc: FlaA1, an inverting 4,6-dehydratase; HP0366, a pyridoxal phosphate (PLP) and Glu-dependent aminotransferase; HP0327, an
acetyl-CoA (AcCoA) utilizing aminotransferase; HP0326b, a hydrolase; HP0178, a synthase that catalyzes the condensation of phosphoenolpyruvate (PEP); and
HP0326a, a synthetase that uses CTP to activate Pse5Ac7Ac.
esis, consistent with its identified role in other SDR enzymes
(30). In addition, we mutated Lys-133 to assess its importance
in catalysis. Both K133M and K133E mutant proteins proved to
lack activity, suggesting that Lys-133 is also important for
enzyme catalysis.
Neither UDP-Glc nor UDP-Gal is a substrate for FlaA1. How-
ever, unlike UDP-Glc, UDP-Gal is able to completely inhibit the
enzyme (11). The crystal structure of FlaA1䡠NADP⫹䡠UDP-Gal
shows that unlike in the complex with UDP-Glc, the hexose ring is
ordered but positioned away from the nicotinamide ring, allowing
it to make specific interactions with the enzyme (Fig. 2B). These
increased points of interaction between UDP-Gal and FlaA1 are
apparently sufficient to result in effective inhibition of the enzyme.
Reaction Catalyzed by FlaA1—FlaA1 has been characterized
previously as a 4,6-dehydratase (11), a bifunctional 4,6-dehy-
dratase-4-reductase (10, 11), and a bifunctional 4,6-dehy-
dratase-5-epimerase (12). The structural data presented here
are incompatible with FlaA1 being a 4,6-dehydratase-4-reduc-
tase, which would convert UDP-GlcNAc to UDP-N-acetyl-␣-
D-quinovosamine. The reason is that such a conversion cannot
be accomplished in a manner that regenerates the NADP⫹
cofactor, mandating replenishment. However, we have shown
that the NADP⫹/NADPH is tightly bound to FlaA1 and can-
not be readily dislodged. Yet, it remains possible that FlaA1
is either a 4,6-dehydratase or a 4,6-dehydratase-5-epimerase.
To address the disparity and contradictions on the reaction
catalyzed, we used NMR spectroscopy to elucidate the chemical
structure of the product of FlaA1 catalysis. Because intermedi-
ates of UDP-saccharide biosynthetic pathways are known to be
unstable during reaction product purification (31), the reaction
catalyzed by FlaA1 was monitored directly in an NMR tube so
that the product could be analyzed in a temporal fashion.
Analysis of the NMR data obtained for the FlaA1 product was
inconsistent with it being UDP-N-acetyl-␣-D-quinovosamine.
Instead, the compound was identified as UDP-2-acetamido-
2,6-dideoxy-␤-L-arabino-hexose-4,4-diol (Supplemental Mate-
AUGUST 25, 2006 • VOLUME 281 • NUMBER 34
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rial). By taking into consideration the aqueous conditions, this
gem-diol compound is in equilibrium with UDP-2-acetamido-2,6-
dideoxy-␤-L-arabino-hex-4-ulose. Therefore, we have identified
FlaA1 to be a UDP-GlcNAc 4,6-dehydratase that additionally
inverts the chirality at the C-5 position. Henceforth we will
describe the activity of FlaA1 as an inverting 4,6-dehydratase activ-
ity, to distinguish it from other 4,6-dehydratases that do not affect
the chirality at C-5. Note that the reaction catalyzed by FlaA1 can
be accomplished in a manner that regenerates the cofactor during
catalysis, consistent with tight binding of NADP⫹/NADPH.
DISCUSSION
Our structural studies have shown that FlaA1 is a hexameric
enzyme belonging to the SDR superfamily, possessing inverting
4,6-dehydratase activity. To our knowledge FlaA1 is the first
SDR superfamily member with a hexameric quaternary struc-
ture and the first inverting 4,6-dehydratase that is structurally
characterized. However, three-dimensional structures of sev-
eral 4,6-dehydratases that retain the chirality of the C-5 atom
have been determined. Specifically, structural studies have
been reported for dTDP-Glc 4,6-dehydratases (29, 32, 33),
GDP-Man 4,6-dehydratases (34, 35), and CDP-Glc 4,6-dehy-
dratases (36, 37). These enzymes share 18 –21% sequence iden-
tity and possess a common structural core of ⬃270 residues. In
comparison, FlaA1 shares only 12–16% sequence identity with
these enzymes, and the structural common core is limited to
⬃220 residues. In fact, FlaA1 is as similar, if not more so, to
4-epimerases such as WbpP as it is to 4,6-dehydratases (11).
Among the different retaining 4,6-dehydratases the dTDP-
Glc 4,6-dehydratase RmlB has become the archetype. A
detailed reaction mechanism has been proposed for RmlB,
which consists of three sequential steps (29, 38, 39). In the first
step, the C-4 hydroxyl is oxidized to form a 4-keto group. This
step employs the conserved (S/T)YK triad, and its mechanism is
identical to what has been described previously for 4-epime-
rases (40). In the second step dehydration occurs across the
JOURNAL OF BIOLOGICAL CHEMISTRY 24493
Structure and Mechanism of Inverting 4,6-Dehydratase FlaA1
bond between C-5 and C-6, thus forming a 4-keto-5,6-ene-hex-
ose. Here the C-5 proton is abstracted by a catalytic base (Glu-
135), whereas the C-6 hydroxyl is simultaneously protonated by
a catalytic acid (Asp-134), effecting a concerted elimination of
water. In the concluding reduction step the hydride from the
nicotinamide cofactor is transferred to C-6, whereas C-5 is re-
protonated, resulting in the formation of a methyl group. The
catalytic acid for this final step has been proposed to be the Tyr
of the (S/T)YK triad or alternatively Glu-135. We propose that
the inverting 4,6-dehydratase reaction mechanism for FlaA1 is
analogous to that described for RmlB: a three-step sequential
mechanism composed of oxidation, intramolecular dehydra-
tion, and reduction, in which the inversion of C-5 chirality is
achieved in the concluding reduction step. Intriguingly,
although much of the FlaA1 reaction mirrors that of RmlB, the
enzyme does not have an analogous constellation of active site
residues; specifically, the structural analog of the catalytic base
in the dehydration step is Lys-133 in FlaA1, whereas it is Glu-
135 in RmlB. Note that the FlaA1 K133E mutant that mimics
the RmlB active site constellation is catalytically inactive.
To assess details of the reaction mechanisms for FlaA1, the
pKa values for the suggested catalytic residues Asp-132, Lys-
133, and Tyr-141 were computed along the reaction coordinate
(Fig. 3). These simulations are in full agreement for the role of
Tyr-141 in the initial oxidation step. However, the calculations
suggested that for the subsequent dehydration step, Asp-132
could not act as a catalytic acid nor could Lys-133 function as a
catalytic base. This finding is in contrast to the roles of analo-
gous residues in the reaction mechanism of RmlB. The com-
puted increase in pKa for Asp-132 after the first oxidation step
suggests that Asp-132 increases the nucleophilic character of
the C-6 oxygen, favoring proton abstraction from Lys-133. In
this scheme, Lys-133 acts sequentially as a catalytic acid proto-
nating the C-6 hydroxyl group and a catalytic base abstracting
the C-5 proton, thus resulting in water elimination. This dual
function for Lys-133 is reminiscent of the proposed role for
Lys-73 in RTEM-1 ␤-lactamase, which is suggested to first
function as a base and then as an acid, effecting the transfer of a
proton from a serine to the ␤-lactam antibiotic (41). As for the
final reduction step, a hydride that is transferred from NADPH
to C-5 and C-6 is protonated, resulting in the change of chirality
at the C-5 center. Our computational analyses suggest that the
catalytic acid for this step is neither Tyr-141 nor Lys-133,
instead that the likely candidate is the water molecule gener-
ated in the previous step.
A time course analysis of the reaction clarifies some of the rea-
sons for the previously erroneous characterization of FlaA1. We
observe that after an hour a second product appears, UDP-2-acet-
amido-2,6-dideoxy-␣-D-xylo-hexose-4,4-diol (see Supplemental
Material). This compound is identical to the product of FlaA1,
except for an inversion of chirality at C-5. This second product is
most likely the result of racemization of the FlaA1 product
through enolization, involving a double bond between C-4 and
C-5. Whether this racemization occurs in the FlaA1 active site or
in solution is unclear. However, given the large time delay for the
appearance of the second product, it is doubtful that there is a
physiological relevance to the racemization reaction.
By having determined the catalytic role of FlaA1 to be an
24494 JOURNAL OF BIOLOGICAL CHEMISTRY
inverting 4,6-dehydratase, the next question to address would
be to determine its role in H. pylori. Recently, several studies
have addressed an intriguing link between the biosyntheses of
several virulence factors and protein glycosylation in H. pylori
(9). In particular, pseudaminic acid (Pse) derivatives such as
5,7-diacetamido-3, 5, 7, 9-tetradeoxy-L-glycero-L-manno-nonu-
losonic acid (Pse5Ac7Ac) have been demonstrated to play an
integral role in the protein glycosylation of H. pylori and closely
related Campylobacter jejuni (10, 12, 42, 43). Pse5Ac7Ac is a
nine-carbon saccharide that has been exclusively found in bac-
teria, and its activated form is as a CMP-saccharide in H. pylori
(10). Based on the recent development in the studies of biosyn-
thesis of sialic acid in bacteria (44, 45) and by anticipating the
plausible catalytic steps to occur in such a pathway, the com-
plete genome sequence of H. pylori (46, 47) was used to identify
candidates in a likely pathway for the biosynthesis of CMP-
Pse5Ac7Ac in H. pylori (Fig. 4). This pathway starts with UDP-
GlcNAc, which is a common precursor in complex saccharide
Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 4, 2015
biosynthesis, and involves six enzymes. To date, biochemical
data are available for the first two enzymes (FlaA1 and HP0366)
(11, 12, 20). The assignment of the remaining enzymes involved
in this proposed pathway was based on sequence similarity of
these enzymes with other proteins possessing similar functions
and by the involvement of their homologs in bacterial protein
glycosylation (42, 48 –50).
In conclusion, the structural studies of FlaA1 have unveiled
the three-dimensional structure and reaction mechanism of an
inverting 4,6-dehydratase involved in the CMP-Pse5Ac7Ac
biosynthetic pathway that is critical for H. pylori pathogenicity.
The fundamental understanding of the reaction mechanism of
FlaA1 involving Lys-133, which plays the dual function during
dehydration, will assist in future efforts to develop antimicro-
bial treatments targeting protein glycosylation in H. pylori and
other related bacterial pathogens.
Protein Data Bank Accession Codes—The atomic coordinates
and structure factors have been deposited with the following
accession codes: 2GN4 (FlaA1䡠NADPH䡠UDP-GlcNAc), 2GN6
(FlaA1䡠 NADP⫹䡠UDP-GlcNAc), 2GN8 (FlaA1䡠 NADP⫹䡠UDP),
2GN9 (FlaA1䡠 NADP⫹䡠UDP-Glc), and 2GNA (FlaA1䡠
NADP⫹䡠UDP-Gal).
Acknowledgments—We thank Bing Xiong for assistance with compu-
tational analyses, Dianne Moyles for assistance in transmission elec-
tron microscopy, and Jean-Robert Brisson, Ian Schoenhofer, and
David McNally for NMR analyses.
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JOURNAL OF BIOLOGICAL CHEMISTRY 24495
Enzyme Catalysis and Regulation:
Structural Studies of FlaA1 from
Helicobacter pylori Reveal the Mechanism
for Inverting 4,6-Dehydratase Activity

Noboru Ishiyama, Carole Creuzenet, Wayne

L. Miller, Melinda Demendi, Erin M.
Anderson, George Harauz, Joseph S. Lam and
Albert M. Berghuis
J. Biol. Chem. 2006, 281:24489-24495.

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doi: 10.1074/jbc.M602393200 originally published online May 1, 2006

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