Characterization of a bifunctional pyranose-furanose mutase from Campylobacter jejuni 11168

Article abstract of DOI:10.1074/jbc.M109.072157

UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-D-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.

Full text of DOI:10.1074/jbc.M109.072157

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 493–501, January 1, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Characterization of a Bifunctional Pyranose-Furanose Mutase
□
S
from Campylobacter jejuni 11168*
Received for publication, October 2, 2009, and in revised form, October 29, 2009 Published, JBC Papers in Press, November 3, 2009, DOI 10.1074/jbc.M109.072157
Myles B. Poulin‡§1, Harald Nothaft§¶, Isabelle Hug§¶, Mario F. Feldman§¶2, Christine M. Szymanski§¶3
and Todd L. Lowary‡§4
,
From the Departments of ‡Chemistry and ¶Biological Sciences and the §Alberta Ingenuity Centre for Carbohydrate Science,
University of Alberta, Edmonton, Alberta T6G 2R3, Canada
UDP-galactopyranose mutases (UGM) are the enzymes re-
sponsible for the synthesis of UDP-galactofuranose (UDP-Galf)
from UDP-galactopyranose (UDP-Galp). The enzyme, encoded
by the glf gene, is present in bacteria, parasites, and fungi that
express Galf in their glycoconjugates. Recently, a UGM homo-
logue encoded by the cj1439 gene has been identified in Campy-
lobacter jejuni 11168, an organism possessing no Galf-contain-
ing glycoconjugates. However, the capsular polysaccharide from
this strain contains a 2-acetamido-2-deoxy-D-galactofuranose
(GalfNAc) moiety. Using an in vitro high performance liquid
chromatography assay and complementation studies, we char-
acterized the activity of this UGM homologue. The enzyme,
which we have renamed UDP-N-acetylgalactopyranose mutase
(UNGM), has relaxed specificity and can use either UDP-Gal
or UDP-GalNAc as a substrate. Complementation studies of
mutase knock-outs in C. jejuni 11168 and Escherichia coli
W3110, the latter containing Galf residues in its lipopolysaccha-
ride, demonstrated that the enzyme recognizes both UDP-Gal
and UDP-GalNAc in vivo. A homology model of UNGM and
site-directed mutagenesis led to the identification of two active
site amino acid residues involved in the recognition of the
UDP-GalNAc substrate. The specificity of UNGM was char-
acterized using a two-substrate co-incubation assay, which
demonstrated, surprisingly, that UDP-Gal is a better sub-
strate than UDP-GalNAc.
4) and is a major structural component of the mycobacterial cell
wall (5). In many pathogenic microorganisms, these Galf resi-
dues are essential for cell viability or play a crucial role in cell
physiology (6, 7). For this reason, and because hexofuranose
sugars are absent in mammalian cell saccharide structures (1),
there has been a surge of interest in studying and identifying
inhibitors of Galf biosynthesis (8).
The sugar nucleotide UDP-Galf is the precursor of Galf and is
incorporated into growing oligosaccharides via galactofurano-
syltransferase-mediated reactions (9). First identified in Esche-
richia coli (10), the enzyme UDP-D-galactopyranose mutase
(UGM) is responsible for the biosynthesis of UDP-Galf via the
ring contraction of UDP-galactopyranose (UDP-Galp). UGM is
encoded by the glf gene for which homologues have since been
identified in Klebsiella pneumoniae (11), mycobacterial species
(12), and in various eukaryotic pathogens (13, 14). Since the
advent of rapid genome sequencing, a number of putative
UGMs have been identified throughout the microbial species;
however, very few of the gene products have been confirmed by
biochemical analysis.
UGM is a flavoprotein and catalyzes the reversible ring con-
traction of UDP-Galp to UDP-Galf via a unique mechanism
(Fig. 1) (15). The noncovalently bound FAD co-factor is directly
involved in catalysis and must be in the reduced form for the
enzyme to be active (16). Because of the interest in UGM as a
drug target (8), significant work has been done to study its
Ϫ
mechanism, and it has been shown that the reduced FADH
In nature, hexose sugars are found predominantly in the
thermodynamically favored pyranose ring form; however, hex-
ose sugars in the furanose ring form are found in bacteria, fungi,
and parasites (1, 2). For example, D-galactofuranose (Galf)5 is a
component in many microbial cell surface oligosaccharides (3,
acts as a nucleophile and displaces the anomeric UDP to form a
covalent intermediate (17). Formation of an iminium ion
breaks the O5–C1 bond of the galactose moiety leading to a
covalently bound acyclic intermediate. This species can then
cyclize to the furanose ring form.
Although the enzyme mechanism is generally understood,
there are still many unanswered questions about the enzyme-
substrate interactions. The UGM protein structure contains a
mobile loop region, which adopts either an open or closed form
in the crystal structures that have been determined to date (15,
18) with the closed structure being the catalytically active form.
This loop has been shown to close upon substrate binding (19),
and a conserved arginine (Arg-174 in K. pneumoniae, Arg-170
in E. coli, and Arg-180 in Mycobacterium tuberculosis) has been
found to be essential for UGM activity (20). This arginine
appears to stabilize the negatively charged diphosphate back-
bone of the sugar nucleotide substrate. Many synthetic ana-
logues (21–26) have been used to probe the mechanism of
UGM and investigate substrate binding, but until recently, no
* This work was supported in part by the Alberta Ingenuity Centre for Carbo-
hydrate Science and National Sciences and Engineering Research Council
of Canada.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S2 and Tables S1–S2.
1 Recipient of a studentship award from Alberta Ingenuity.
2 Alberta Heritage Foundation for Medical Research Scholar.
3 Alberta Ingenuity Scholar. To whom correspondence may be addressed.
Tel.: 780-248-1234; Fax: 780-492-9234; E-mail: cszymans@ualberta.ca.
4 To whom correspondence may be addressed. Tel.: 780-492-1861; Fax: 780-
492-7705; E-mail: tlowary@ualberta.ca.
5 The abbreviations used are: Galf, D-galactofuranose; Cj, Campylobacter
jejuni; CPS, capsular polysaccharide; Ec, Escherichia coli; LPS, lipopolysac-
charide; UGM, UDP-galactopyranose mutase; UNGM, UDP-N-acetylgalac-
topyranose mutase; Galp, galactopyranose; HPLC, high pressure liquid
chromatography.
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 493

Bifunctional Pyranose-Furanose Mutase from C. jejuni
GalfNAc from UDP-GalpNAc (33). Herein, we report studies
on the protein produced by expression of cj1439 and demon-
strate its activity as a UDP-N-acetylgalactopyranose mutase
(UNGM). We also have demonstrated that the enzyme can use
both UDP-Gal and UDP-GalNAc as substrates and have inves-
tigated the origins of this substrate selectivity using site-di-
rected mutagenesis to identify key residues that allow for the
turnover of UDP-GalNAc.
EXPERIMENTAL PROCEDURES
Preparation of UDP-sugars—UDP-Galp and UDP-GalpNAc
were obtained from Aldrich and used without further purifica-
tion. UDP-Galf was prepared from synthetic Galf-1-phosphate
(38) using galactose-1-phosphate uridyltransferase and UDP-
glucose pyrophosphorylase as described by Errey et al. (39). All
stock solutions were prepared by dissolving the appropriate
quantity of UDP-sugar in 100 mM potassium phosphate (pH
7.4). Before use, stock solutions were calibrated by HPLC co-
injection with a known concentration of UDP.
FIGURE 1. UGM reaction mechanism.
Cloning, Expression, and Purification of Glf Proteins—For
MFF1 (Ec-glf mutant) and Cj1439c (Cj-glf mutant) comple-
mentation and in vivo Glf analyses, glf alleles were put under the
control of the constitutive C. jejuni-E. coli shuttle promoter
replacing the gfp gene on plasmid pWM1007 (40). Cj-glf alleles
FIGURE 2. Capsular polysaccharide structure of C. jejuni 11168. Shown is
the tetrasaccharide repeat unit of C. jejuni 11168 (HS:2 serotype) with the
GalfNAc residue shown in boldface.
ligand-bound crystal structures have been available. Trypto- were amplified by PCR using oligonucleotides CS261 and
phan fluorescence (15) and molecular modeling have pre- CS262 that introduce restriction sites for EcoRI and BsrGI,
dicted that the uridine of the UDP-Gal base stacks with Trp- whereas Ec-glf was amplified with oligonucleotides CS362 and
160 (numbering for K. pneumoniae) (27); in contrast, recent CS363 introducing an EcoRI site in the 5Ј end of Ec-glf. For both
crystal structures of the K. pneumoniae UGM with bound species, a C-terminal His tag was introduced via PCR, where the
UDP-Glcp (28) and UDP-Galp (29) show that the uridine plasmid pJHCV32 (41) and chromosomal DNA of C. jejuni
stacks against tyrosine 155 in the active site. This discrep- 11168-V26 (42) served as template DNA, respectively. The
ancy demonstrates that many of the key binding interactions EcoRI-BsrGI-digested Cj-glf PCR product was ligated with the
responsible for the substrate specificity of UGM still remain purified 8643-bp pWM1007 vector DNA fragment obtained
to be elucidated.
after digestion with the same enzymes. The Ec-glf PCR product,
Although Galf is the most common naturally occurring subsequently treated with T4 DNA polymerase and EcoRI, was
hexofuranose, it is not unique. 6-Deoxy-D-galactofuranose (30), inserted into the purified 8297-bp pWM1007 vector subse-
6-deoxy-L-altrofuranose (31), and 2-acetamido-2-deoxy-D-ga- quently treated with SfuI, T4 DNA polymerase, and EcoRI. For
lactofuranose (32, 33), among others, have also been identified expression in C. jejuni, the kan (kanamycin) cassette within the
in bacterial saccharide structures. However, little is known pWM1007-Cj-glf construct was replaced by the cat (chloram-
about the biosynthesis of these other hexofuranose sugars. Pre- phenicol) cassette after EcoRV digestion of the vector and liga-
vious work has established that the UGM from K. pneumoniae, tion with an 842-bp DNA fragment containing the cat cassette
which has been the most studied, is unable to catalyze the syn- isolated from plasmid pRY109 (43) after SmaI digestion. A sim-
thesis of either UDP-Fucf or UDP-GalfNAc (26, 34). Recently it ilar strategy was carried out for the pWM1007 Ec-glf con-
has been shown that a homologue of the glf gene, fcf2 in E. coli structs, except that the 8428-bp vector fragment was purified
O52, acts as a Fucf mutase enzyme for the biosynthesis of TDP- after partial digestion with EcoRV. Orientation of the cat gene
Fucf (35). This protein has 60% identity to the K. pneumoniae on the resulting plasmids (same orientation as the nonpolar kan
UGM, but the origin of the difference in substrate tolerance is cassette) was verified by restriction analyses. For high yield
unknown.
expression of Ec-glf, the corresponding gene was amplified by
The bacterium Campylobacter jejuni is a foodborne patho- PCR using oligonucleotides CS372 and CS373 to introduce
gen that is a leading cause of diarrheal disease worldwide (36). NdeI and XhoI restriction sites, respectively. Plasmid pJHCV32
Infections by this organism have also been linked to the devel- (41) served as template DNA for the PCR. After restriction
opment of the neurological disorder Guillain-Barre´ syndrome digestion, the purified DNA fragment was ligated into plasmid
(37). Previous work showed that the capsular polysaccharide pET22b cut with the same enzymes.
(CPS) from the 11168 strain contains a GalfNAc residue (Fig. 2)
Expression of soluble C-terminal hexahistidine-tagged
(33). C. jejuni 11168 also contains a homologue of the glf gene E. coli UGM protein from plasmid pET-Ec-glf in BL21 was
cj1439. Because no Galf residues have been found in C. jejuni observed after induction with 0.01 mM isopropyl ␤-D-1-thioga-
11168 glycoconjugates, it has been proposed that the cj1439 lactopyranoside for 2 h at room temperature (22 °C). C-termi-
gene product is responsible for the biosynthesis of UDP- nal hexahistidine-tagged C. jejuni UNGM was expressed in
494 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Bifunctional Pyranose-Furanose Mutase from C. jejuni
E. coli DH5␣ from plasmid pWM1007 (kan)-Cj-glf after growth tion of the appropriate peaks from the HPLC. The ratio was
for 18 h at 28 °C. Soluble UNGM-His6 proteins were purified determined for both UDP-Gal and UDP-GalNAc substrates in
by nickel-nitrilotriacetic acid affinity chromatography as de- both the forward and reverse directions. For the forward reac-
scribed previously.
tion, UDP-Galp or UDP-GalpNAc (1 mM) was incubated with
UNGM Activity Assay—The activity of purified wild-type the appropriate mutase protein (3.9 ␮M) in 30 ␮l of 100 mM
and mutant proteins was assayed by incubating a mixture of potassium phosphate buffer (pH 7.4) with freshly prepared
sugar nucleotide (UDP-Galp, UDP-Galf, UDP-GalpNAc, or sodium dithionite (20 mM) until equilibrium was reached as
UDP-GalfNAc, 1 mM) and mutase protein (3.9 ␮M) in 30 ␮l of indicated by a constant product/substrate ratio by HPLC. The
100 mM potassium phosphate buffer (pH 7.4) containing freshly same procedure was used for the reverse reaction but instead
prepared sodium dithionite (20 mM) for 2-, 5-, 10-, and 20-min using UDP-Galf or UDP-GalfNAc (1 mM) as the starting
periods at 37 °C. Reactions were monitored by HPLC (Varian substrate.
Prostar 210) following conditions similar to those previously
Complementation of C. jejuni 11168 glf Knock-out—Shuttle
reported by Zhang and Liu (16). A C18 column (Microsorb-MV, plasmids (pWM1007/cat derivatives) expressing the E. coli glf
Varian, 4.6 ϫ 250 mm) was used, and elution was done with 50 and C. jejuni glf genes were mobilized into C. jejuni 11168 wild-
mM triethylammonium acetate buffer (pH 6.5) containing 1.5% type and the glf mutant as described (44). Capsular polysaccha-
acetonitrile. A flow rate lower than that described earlier (0.6 rides of the resulting strains were prepared as described (45)
ml/min) (16) was used to increase separation, and the UV and visualized after 16.5% deoxycholate PAGE by silver staining
detector was set to a wavelength of 262 nm. Base-line resolution using the protocol of Tsai and Frasch (46) with the modification
for all substrates was achieved, and the retention times for that fixing was performed for only 2 h.
UDP-Galp, UDP-Galf, UDP-GalpNAc, and UDP-GalfNAc
UGM Activity in E. coli—The E. coli glf mutant strain MFF1
were found to be 8.8, 10.6, 10.0, and 12.5 min, respectively. The (47) was transformed with plasmids containing E. coli wbbL
amount of conversion was determined by integration of the (pMF19 (47)) and either empty vector pWM1007 or pWM1007
product and starting material peaks.
containing glf from E. coli or from C. jejuni. Strains were grown
Enzymatic Synthesis of UDP-GalfNAc (Compound 2)—To overnight after induction with 0.5 mM isopropyl ␤-D-1-thioga-
confirm the identity of the product of the UNGM incubation lactopyranoside. Lipopolysaccharides (LPS) were extracted fol-
with UDP-GalpNAc, a milligram scale reaction of UDP- lowing a modified protocol of Marolda et al. (48). Briefly, cells
GalfNAc was carried out. UDP-GalpNAc (1 mM, 25 mg) was were adjusted to an absorbance of 3.0 (600 nm wavelength),
incubated with 4.7 ␮M UNGM in 2 ml of 100 mM potassium resuspended in 150 ␮l of lysis buffer (2% SDS, 4% ␤-mercapto-
phosphate buffer (pH 7.4) containing freshly prepared sodium ethanol, 10% glycerol, 0.5 M Tris (pH 6.8)), and incubated at
dithionite (20 mM) for 1 h at 37 °C. Compound 2 was purified by 100 °C for 10 min. After addition of 2 ␮l of proteinase K (20
HPLC using a C18 column (Microsorb, Varian, 21.4 ϫ 250 mm). mg/ml), samples were incubated for 2 h at 60 °C. To this solu-
The eluent used was 50 mM triethylammonium acetate (pH 6.5) tion was added 150 ␮l of hot phenol, and samples were incu-
containing 1.5% acetonitrile at a flow rate of 7.0 ml/min. The bated at 70 °C for 15 min, followed by 10 min on ice. After
retention time of compound 2 was 22.3 min under these con- centrifugation, the aqueous phase was mixed with 250 ␮l of
ditions. The product was further purified by chromatography EtOH and centrifuged, and the precipitated LPS were dried at
on a Sephadex G-15 column (21.4 ϫ 250 mm) eluting with Milli room temperature. LPS corresponding to an absorbance (A600
)
Q H2O. The product was lyophilized to obtain a white powder of 0.45 were separated by SDS-PAGE, and LPS silver staining
(1 mg, 4% yield). The 1H NMR data for the product can be found was performed according to Tsai and Frasch (46).
in the supplemental Table S2. Negative ion high resolution
Homology Model of C. jejuni UNGM with Bound
electrospray ionization mass spectrometry m/z calculated for UDP-GalfNAc—A homology model of C. jejuni 11168 UNGM
C17H25N3O17P2 [M Ϫ 2H]2Ϫ was 302.5335 and found was was generated based on the crystal structure of the E. coli UGM
302.5339.
in the closed ring form (Protein Data Bank code 1I8T, chain A)
NMR Spectroscopy—NMR spectra were obtained at 27 °C (15) using ESyPred3D (49). Chain A was used as it has the
using a Varian DirectDrive two-channel spectrometer operat- mobile loop region in the active “closed” conformation. Using
ing at 499.822 MHz for 1H. Samples were prepared at concen- Autodock 4.0 (50), the UDP-GalpNAc and UDP-GalfNAc sub-
trations of ϳ1.5 mM in D2O. Chemical shifts were externally strates were modeled into the active site. For each substrate, the
referenced to 0.1% acetone signal (2.225 ppm). A sweep width low energy conformation, in which the nucleotide conforma-
of 6010 Hz was used for measurement of the one-dimensional tion agreed with the published crystal structure data of UGM
1H NMR spectrum with a 3.0 s relaxation time. The free induc- bound to UDP-Glcp (28) and UDP-Galp (29), was chosen to
tion decay signal gave a final digital resolution of 0.1 Hz/point. represent a plausible binding mode.
All coupling constants are reported in hertz (see supplemental
Mutagenesis of C. jejuni UNGM—The R59H, R168K, and
Table S2). A two-dimensional 1H–1H COSY spectrum of com- R59H/R168K mutants were prepared using the QuikChange
pound 2 (supplemental Fig. S1) was obtained using a 6010 Hz XL II protocol by Stratagene (51). In the case of the R59H and
sweep width with a 1.0-s acquisition time, a 0.1 relaxation delay, R168K, mutants were prepared from the pWM1007 plasmid
and 426 t1 increments.
containing the glf gene isolated from E. coli DH5␣ cells using
Determination of the Pyranose-Furanose Distribution at R59H_F and R59H_R or R168K_F and R168K_R (supplemental
Equilibrium—The distribution of pyranose to furanose ring Table S1) as primer pairs, respectively. In the case of R59H/
forms was measured at equilibrium and determined by integra- R168K, the double mutant was prepared using the R59H
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 495

Bifunctional Pyranose-Furanose Mutase from C. jejuni
tion time of 11.2 min. The incuba-
tion mixtures were monitored by
HPLC as described above. In all
cases, a concentration of protein
was used so that less than 40% con-
version to the pyranose product was
observed. The concentrations of
UDP-Galp or UDP-GalpNAc were
determined by integration of the
appropriate peaks on the HPLC
trace, and these were used to deter-
mine the initial velocities. Each
assay was performed in duplicate for
all proteins for each of the furanose
substrates. The calculated enzyme
specificity was determined using a
modification of the Michaelis-Men-
ten Equation 1 for two competing
substrates (52),
FIGURE 3. Functional characterization of C. jejuni UNGM. A, with UDP-GalNAc as the substrate. Trace i,
retention time for a standard solution of UDP-GalpNAc. Trace ii, incubation of UDP-GalpNAc with C. jejuni
UNGM at equilibrium. Trace iii, standard solution of the purified product (UDP-GalfNAc) from the reaction of
UDP-GalpNAc with C. jejuni UNGM. Trace iv, incubation of UDP-GalfNAc with C. jejuni UNGM at equilibrium. The
same ratio of 93:7 UDP-GalpNAc to UDP-GalfNAc is seen as in trace ii. B, with UDP-Gal as the substrate. Trace i,
retention time for a standard solution of UDP-Galp. Trace ii, incubation of UDP-Galp with UNGM at equilibrium.
Trace iii, standard solution of UDP-Galf produced enzymatically from Galf-1-phosphate. Trace iv, incubation of
UDP-Galf with C. jejuni UNGM at equilibrium. The same ratio of 93:7 UDP-Galp to UDP-Galf seen in trace ii is also
observed. The peak at ϳ8 min corresponds to UMP.
vA ͑kcat/KM͒ ͓A͔
A
ϭ
(Eq. 1)
vB ͑kcat/KM͒ ͓B͔
B
RESULTS
C. jejuni UNGM Functions as a
UNGM in Vitro—When annotated,
the glf gene product from C. jejuni
11168 was proposed to be a UGM based on homology (53),
despite the lack of Galf residues its glycoconjugates. Although it
was later suggested that Glf may function in biosynthesis of
GalfNAc in the CPS (33), the function of the protein has never
been examined. In this study, the purified C. jejuni UNGM was
examined by incubation with UDP-GalpNAc (compound 1)
under the reducing conditions modified from Liu and Zhang
(16), and conversion to products was monitored by reverse
phase HPLC. Formation of a longer retention time product
(12.5 min) was observed, and at equilibrium, a ratio of 7:93 with
respect to the UDP-GalpNAc peak resulted, as seen in Fig. 3A
(traces i and ii). This reflects the equilibrium ratios observed for
the K. pneumoniae (26) and E. coli (16) UGM with UDP-Gal.
Scaling up the reaction gave access to sufficient quantities of the
reaction product to characterize by 1H NMR spectroscopy (Fig.
4B) and mass spectrometry. The observed mass was consistent
with the product being UDP-GalfNAc (compound 2), and the
mutant pWM1007 plasmid and the R168K_F and R168K_R
primer pair. After mutagenesis, the plasmid DNA was isolated
from the XL10-gold cells and sequenced before being trans-
formed into DH5␣ cells for protein expression.
Competitive Substrate Specificity Assay—A mixture of UDP-
Galf (0.5 mM) and UDP-GalfNAc (0.5 mM) was incubated with
the appropriate mutase protein (3.9 ␮M) in 100 mM potassium
phosphate buffer (pH 7.4) containing freshly prepared sodium
dithionite (20 mM) for 5 min. The reactions were again moni-
tored using the same modified HPLC conditions of Zhang and
Liu (16). In this case, the use of a lower concentration of aceto-
nitrile (1.25%) and a flow rate of 0.6 ml/min allowed for better
resolution of all four product and substrate peaks (UDP-Galp,
UDP-Galf, UDP-GalpNAc, and UDP-GalfNAc). Using these
conditions, these compounds were found to have retention
times of 10.0, 12.4, 11.6, and 15.4 min, respectively. The relative
specificity was determined by integration of the UDP-Galp and
UDP-GalpNAc peaks.
1
resonances in the H NMR spectrum closely matched those
previously reported for GalfNAc residues in the CPS of C. jejuni
11168 (33) and the O-specific polysaccharide of Proteus penneri
strain 22 (32). In particular, the characteristic coupling pattern
for the GalfNAc H-4 proton and the upfield shift of the H-5
proton confirmed a galactofuranose ring configuration (11).
Incubation of the isolated product 2 with UNGM under the
same reducing conditions gave, as expected, an equilibrium
ratio of 7:93 UDP-GalfNAc to UDP-GalpNAc (Fig. 3B).
Determination of UNGM and Mutant Kinetic Parameters—
Kinetic parameters for wild-type C. jejuni UNGM and each of
the mutants were determined following a kinetic assay modi-
fied from the procedure reported by Zhang and Liu (16). Reac-
tions were prepared containing an appropriate concentration
of the desired protein with UDP-Galf or UDP-GalfNAc (10,
12.5, 25, 50, 100, 250, and 500 ␮M) in a final volume of 60 ␮l of
100 mM potassium phosphate (pH 7.4) containing 20 mM of
freshly prepared sodium dithionite. Incubations were carried
out for 5 min at 37 °C and then promptly quenched by heating
E. coli UGM Does Not Interconvert UDP-GalpNAc and
UDP-GalfNAc—Earlier work established that the UGM of K.
to 90 °C for 5 min. The sugar nucleotides do not decompose by pneumoniae does not interconvert UDP-GalpNAc and UDP-
loss of UDP under these quenching conditions, as evidenced by GalfNAc (26), but no published data were available for the
the lack of a peak in these HPLCs for UDP, which has a reten- E. coli UGM. The E. coli UGM has a higher sequence identity,
496 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Bifunctional Pyranose-Furanose Mutase from C. jejuni
the protein was incubated with both
UDP-Galp (compound 3) and UDP-
Galf (compound 4) (39). Intercon-
version between compounds 3 and 4
was observed in both cases (Fig. 3B).
As was observed for compound 2,
the equilibrium ratio was consistent
with the 7:93 UDP-Galf to UDP-
Galp ratio reported previously (16).
In a second experiment, co-in-
cubation of UNGM with a 50:50
mixture of UDP-Galf and UDP-
GalfNAc was carried out. This
experiment was intended to provide
an estimate of the relative specificity
of the enzyme for these two sub-
FIGURE 4. 1H NMR analysis of the product of C. jejuni UNGM reaction with UDP-GalpNAc. A, region of the
1H NMR spectrum of the starting material UDP-GalpNAc. B, same region of the 1H NMR spectrum for the UNGM strates. At equilibrium, the ratio of
reaction product. The observed chemical shifts and coupling constants are consistent for UDP-GalNAc in the
furanose ring form.
the corresponding pyranose prod-
uct was 2.48:1 in favor of UDP-Galp
as shown in Fig. 7.
TABLE 1
C. jejuni UNGM Has UGM Activity in E. coli—Because the
Kinetic parameters for the C. jejuni UNGM and mutants
specificity of UNGM allows it to act as a UGM in vitro, we
UDP-Gal:
desired a method to test this activity in a cellular environment
Enzyme
Substrate
Km
kcat
kcat/Km
UDP-GalNAc
similar to the ⌬glf complementation experiment in C. jejuni.
Unfortunately, no C. jejuni serotypes containing Galf residues
have been identified. Conveniently, common E. coli laboratory
strains such as W3110 are derived from serogroup O16, which
contains Galf as a component of the LPS O-antigen. In this
species, the O-antigen is composed of a pentasaccharide
repeating unit of (132)-␤-D-Galf-(136)-␣-D-Glcp-(l33)-␣-L-
Rhap-(133)-[(136)-␣-D-Glcp]-␣-D-GlcpNAc (47). The W3110
strain does not display a smooth LPS phenotype, due to an
Ϫ1
Ϫ1
␮M
min
␮MϪ1 min
Wild type
UNGM
R59H
UDP-Galf
UDP-GalfNAc
UDP-Galf
45 Ϯ 3
40 Ϯ 6
178 Ϯ 4
4.1
1.5
15
114 Ϯ 5
2.8
92 Ϯ 20 590 Ϯ 50
6.4
UDP-GalfNAc
UDP-Galf
61 Ϯ 7
65 Ϯ 9
26 Ϯ 1
96 Ϯ 4
0.43
1.5
R168K
3.8
21
UDP-GalfNAc 231 Ϯ 40
89 Ϯ 7
0.39
4.9
R59H/R168K
UDP-Galf
77 Ϯ 9
59 Ϯ 6
NDa
380 Ϯ 20
13.8 Ϯ 0.5
NDa
UDP-GalfNAc
0.23
E. coli UGM UDP-GalfNAc
NDa
NDa
a ND means none detected.
60%, with the C. jejuni 11168 UNGM than with the K. pneu- insertion sequence disrupting a gene encoding the rhamno-
moniae UGM (38% identity) with which it shares a function. syltransferase (wbbL), which is required for the attachment of
Therefore, we wanted to determine whether the E. coli UGM the second sugar of the O-antigen subunits. Re-introduction of
recognized UDP-GalpNAc as a substrate. We found that no the wbbL gene on a plasmid restores smooth LPS biosynthesis
conversion of UDP-GalpNAc was detected using the E. coli (54, 55). By mutating the E. coli O16 glf gene in a wbbL-comple-
UGM (Table 1), even upon prolonged incubations (60 min). A mented strain, only one incomplete O chain subunit, devoid of
similar experiment, using UDP-GalfNAc as the substrate, also Galf, is attached to the lipid A core (Fig. 5B, lane 2) (47). Wild-
demonstrated no conversion. To ensure that the protein was type LPS production is restored when E. coli glf is reintroduced
active, both UDP-Galp and UDP-Galf were used in the assay, (Fig. 5B, lane 3) (47). To prove in vivo UGM activity of the C.
and activity levels consistent with previously published results jejuni UNGM, we repeated the complementation of the E. coli
(16) were observed.
⌬glf mutation using a plasmid carrying C. jejuni glf. As shown in
C. jejuni CPS Production Is Restored by Expressing the cj1439c the 4th lane of Fig. 5B, the resulting strain produced full-length
Allele in Trans—To test the activity of the glf-His allele in vivo, LPS, indicating that intact O chain subunits containing Galf
the corresponding proteins were expressed in trans in C. jejuni were synthesized and polymerized. Incorporation of GalfNAc
wild-type and the glf mutant strain (Fig. 5A). CPS formation was into the O chain can be ruled out because subunits of the O16
followed by silver staining of crude CPS preparations. Expres- LPS are connected via an ␣-(132) linkage to Galf, which would
sion of the Cj-glf-His allele resulted in restoration of CPS in the be prevented by the presence of an acetamido group at position
cj1439c(glf) mutant, whereas no CPS formation was observed 2, as in GalfNAc. Furthermore, to the best of our knowledge,
upon expression of Ec-glf-His. The expression of an additional serogroup O16 E. coli strains do not produce UDP-GalpNAc.
copy of Cj-glf-His, Ec-glf-His, or the presence of the empty plas- Thus, the E. coli complementation studies indicate that C.
mid (pWM1007/cat, negative control) in C. jejuni 11168 cells jejuni UNGM can interconvert UDP-Galf and UDP-Galp
did not affect CPS formation.
in vivo.
Modeling the Active Site of UNGM Suggests the Origin of
C. jejuni UNGM Interconverts UDP-Galf and UDP-Galp in
Vitro—The natural CPS of C. jejuni 11168 contains only UDP-GalNAc Recognition—To investigate the origin of the
GalfNAc residues and no Galf residues (33). To probe whether increased substrate scope of C. jejuni UNGM compared with
this observation is due to the specificity of the C. jejuni UNGM, the highly homologous E. coli UGM, we examined differences
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 497

Bifunctional Pyranose-Furanose Mutase from C. jejuni
in the active site residues. No crystal structure for the C. jejuni to UDP-Gal (28), which was recently reported. As was seen in
UNGM has been determined; therefore, a homology model was the K. pneumoniae structure, a key base stacking interaction is
generated based on the E. coli UGM for which a structure is predicted to occur with a tyrosine residue (Tyr-150).
available (15). After building the protein model, UDP-GalpNAc
Active site residues are highly conserved between the three
(Fig. 6A) and UDP-GalfNAc (Fig. 6B) were docked into the bacterial UGM for which x-ray structures have been deter-
active site. Inspection of the resulting structure showed that the mined (E. coli, K. pneumonia, and M. tuberculosis) and the C.
nucleotide portion was bound in a similar conformation to that jejuni UNGM. Only one residue in the UNGM active site, argi-
observed in the crystal structure of K. pneumoniae UGM bound nine 59, differed from the conserved histidine found in all three
bacterial UGMs (supplemental Fig. S2). A second residue, argi-
nine 168 in UNGM, differs from the conserved lysine found in
the E. coli and K. pneumoniae UGM. As seen in Fig. 6A, Arg-59
appears to be in close proximity to the carbonyl oxygen of the
acetamido moiety of UDP-GalpNAc. The other residue, Arg-
168, is located adjacent to the conserved arginine, in this case
Arg-169, in the mobile loop region that has previously been
shown to be essential for UGM activity (20).
Mutagenesis of Arginines 59 and 168 Reduces the Ability of
UNGM to Catalyze Interconversion of UDP-GalfNAc to
UDP-GalpNAc—The two active site arginine residues, Arg-59
and -168, were examined for their role in the interconversion of
UDP-GalfNAc and UDP-GalpNAc by UNGM. Site-directed
mutagenesis was used to convert these residues to the corre-
sponding amino acid from the E. coli/K. pneumoniae UGMs,
i.e. arginine 59 to histidine and arginine 168 to lysine. The dou-
FIGURE5.C.jejuniglfgenecomplementsCPSinC.jejuni⌬glfstrainandLPSin
E. coli ⌬glf strain. A, separation of C. jejuni 11168 crude CPS preparations by
16.5% deoxycholate PAGE. Equivalent amounts of sample were loaded in each
ble mutant was also constructed with both R59H and R168K
mutations. The specificity of the mutant UNGM was deter-
lane that originated from bacterial cell cultures adjusted to an absorbance (A600
)
mined using a co-incubation assay with the same 50:50 mixture
of UDP-Galf and UDP-GalfNAc. Again, the ratio of the corre-
sponding pyranose products was used to determine the relative
selectivity of each mutant for the two substrates (Fig. 7). The
R59H mutation resulted in a decrease in the conversion of
UDP-GalfNAc and the selectivity for the corresponding sub-
strate, whereas UDP-Galf conversion increased by greater than
10-fold over the wild-type UNGM. The R168K mutation had a
less pronounced effect causing only an approximate 2-fold
increase in the selectivity for UDP-Galf compared with the
wild-type enzyme. Mutation of both residues R59H and R168K
resulted in the largest decrease in
of 3.0. Silver staining showed formation of CPS in the Cj-wt strain as well as Cj-wt
with pMW1007/cat, pMW1007/Cj-glf, or pMW1007/Ec-glf plasmids. wt is wild
type The C. jejuni 1439c (glf) knock-out strain (Cj-⌬) and the Cj-⌬ strain with
pMW1007/cat plasmid showed no CPS formation. Cj-⌬ strain complemented
with pMW1007/Cj-glf showed restoration of CPS production, and no CPS was
observedinCj-⌬complementedwithpMW1007/Ec-glf.Inallstrainslipooligosac-
charide (LOS) formation was not affected. B, LPS of E. coli strains derived from
MFF1 were extracted after overnight growth and separated by SDS-PAGE. Equiv-
alent amounts of sample were loaded in each lane that originated from bacterial
cell cultures adjusted to an A600 of 0.45. Silver staining shows the production of a
fast migrating band composed of lipid A core plus a GlcNAc residue (wbbL Ϫ
,
glf Ϫ); a band of higher molecular weight due to addition of an incomplete O
antigen subunit to the lipid A core, which is only deficient of the Galf residue
(wbbL ϩ, glf Ϫ); and smooth LPS when both wbbL and either the E. coli or the C.
jejuni glf are present (wbbL ϩ, Ec-glf and wbbL ϩ, Cj-glf), respectively.
the conversion of UDP-GalfNAc to
UDP-GalpNAc and a selectivity
increase for UDP-Galf of greater
than 12-fold in comparison to the
wild-type UNGM.
Analysis of C. jejuni UNGM and
Mutant Kinetics with UDP-Galf and
UDP-GalfNAc Supports Results of
the Co-incubation Assay—At equi-
librium, the pyranose ring form is
favored in the reaction of pyranose-
furanose mutases. Therefore, sub-
strate kinetics are often measured
starting with the furanose form and
FIGURE 6. Homology model of the active site of C. jejuni UNGM. A, homology model of C. jejuni UNGM with
theUDP-GalpNAcsubstratedocked. TheUDP-GalpNAcappearstobeboundinanactiveconformationwiththe
GalpNAc C-1 located in proximity to the FADHϪ cofactor. UDP is positioned similar to that in the crystal
structure of K. pneumoniae bound to UDP-Glc by Kiessling and co-workers (28). Nonconserved active site
residues Arg-59 and Arg-168 and their distance from the acetamido carbonyl oxygen are highlighted.
B, homology model of C. jejuni UNGM with the UDP-GalfNAc substrate bound. The UDP-GalfNAc appear bound
in an inactive conformation (no possible interaction between GalfNAc C-1 and FADHϪ cofactor). Noncon-
served active site residues Arg-59 and Arg-168 are highlighted.
monitoring the formation of the
thermodynamically favored pyr-
anose isomer (56). Thus, the kinetic
parameters were determined for
UNGM and each of the mutants
using UDP-GalfNAc and UDP-Galf
498 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Bifunctional Pyranose-Furanose Mutase from C. jejuni
UDP-GalfNAc. This enzyme, which bears a high sequence
similarity to known UGMs, produces UDP-GalfNAc from
UDP-GalpNAc presumably via a similar ring contraction
mechanism. Both in vitro investigations and an in vivo comple-
mentation experiment support the role of UNGM in the bio-
synthesis of the GalfNAc in C. jejuni 11168. In addition, we have
demonstrated that the C. jejuni UNGM has dual substrate spec-
ificity and can interconvert the furanose and pyranose isomers
of both UDP-Gal and UDP-GalNAc. Furthermore, the UGM
activity of the C. jejuni enzyme has been demonstrated in vivo
where it is able to complement the activity of the E. coli UGM in
a glf gene knock-out.
It is not unusual that bacteria with compact genomes express
enzymes that exhibit more than one activity. These bifunctional
enzymes are widespread among bacteria and allow for the syn-
thesis of many complex structures advantageous to the survival
of the organism while still maintaining a small genome size. An
example is the wbbO gene product from K. pneumoniae, a
galactosyltransferase that catalyzes the transfer of both Galp
and Galf residues in the biosynthesis of the lipopolysaccharide
O1 antigen (58). Bifunctional enzymes have also been charac-
terized in C. jejuni. For example, a single UDP-GlcNAc/Glc
4-epimerase was shown to be involved in the biosynthesis of
three cell surface glycoconjugates in strain 11168 (59). Because
the CPS structures in C. jejuni are highly variable between sero-
types, it is reasonable to hypothesize that a bifunctional UNGM
would be advantageous. However, no Galf residues have been
identified in any glycoconjugate from this organism. Thus, the
inclusion of GalfNAc, instead of Galf, into the CPS appears to be
due to the specificity of the cognate glycosyltransferase.
Comparing Calculated Specificity of C. jejuni UNGM to the
Experimentally Determined Specificity—In this study, two
methods were used to determine the substrate specificity of the
C. jejuni UNGM. The first was the direct determination from
the ratio of product conversion observed in the co-incubation
assay (Fig. 7). The second was calculated using the observed
kinetic parameters for each protein with UDP-Galf or UDP-
GalfNAc (Table 1). The observed trend of substrate specificity
is the same by either method; however, the calculated specific-
ity in each case is lower than that determined using the co-
FIGURE 7. Co-incubation assay of C. jejuni UNGM with 50:50 UDP-GalfNAc
and UDP-Gaf. After co-incubation, the relative amounts of UDP-Galp and
UDP-GalpNAc were determined based on the total amount of products
observed. Shown are the average of three co-incubation assays for each of
the C. jejuni UNGM and the R59H, R168K, and R59H/R168K UNGM mutants.
Also shown is the specificity of the protein represented by the ratio of UDP-
Galp to UDP-GalpNAc.
as substrates. As seen in Table 1, the Km value of wild-type
UNGM is approximately the same for both UDP-Galf and
UDP-GalfNAc; however, the kcat value is larger for UDP-Galf.
The R59H mutant showed only small changes in the Km value
for both substrates as compared with the wild-type UNGM. At
the same time, there is a significant decrease in kcat values
observed for UDP-GalfNAc in addition to an increase in the kcat
values for UDP-Galf. Conversely, the R168K mutant displayed
approximately the same kcat value for both substrates, but the
Km value was significantly larger for UDP-GalfNAc as the sub-
strate. For each protein, the first-order rate constants were
approximated by calculating kcat/Km values for each substrate
(Table 1). The ratio of the first-order rate constants for UDP-
Galf over UDP-GalfNAc could be used to approximate the
specificity of each protein for UDP-Galf as the incubation times
for each substrate remained constant (52). The calculated spec-
ificities in each case mirror those determined using the co-in-
cubation assay (Fig. 7), but the former approach underestimates
these differences.
DISCUSSION
In this paper, we have tested a previously proposed hypoth- incubation assay. The calculated substrate specificity may not
esis (57) that the C. jejuni 11168 gene cj1439 encodes a protein accurately represent the observed specificity as the combined
responsible for the biosynthesis of the UDP-GalfNAc. Our rate of the two competing reactions may be greater than, equal
investigations have shown that, unlike the highly homologous to, or lower than the rate of each individual reaction (60).
UGM from E. coli and K. pneumoniae, the C. jejuni enzyme is Because of this, it appears that the co-incubation assay more
able to convert the sugar nucleotide UDP-GalpNAc to UDP- accurately represents the substrate specificity of the UNGM for
GalfNAc, the precursor of GalfNAc in the CPS. Thus, the pro- its two competing substrates UDP-Galf and UDP-GalfNAc.
tein is a UNGM. In addition, we have demonstrated that the C.
Effect of Two Active Site Arginines on UDP-GalNAc Recogni-
jejuni UNGM also converts UDP-Galp to UDP-Galf both in tion by UNGM—We believed that residues in, or in proximity
vitro and in vivo in E. coli. We also identified two amino acids in to, the active site would play a role in the different substrate
the active site of UNGM that play a role in the interconversion specificity of the C. jejuni UNGM compared with other known
of UDP-GalfNAc and UDP-GalpNAc. Our co-incubation assay UGM enzymes. We also rationalized that these key residues
allowed us to examine, in a single reaction, the substrate spec- would be conserved in the other UGM but not in the C. jejuni
ificity of wild-type UNGM protein and each of the UNGM enzyme. The only two residues that fit these criteria were
mutants.
Arg-59 and Arg-168. In both cases, these residues were found to
Cj1439 Encodes for a Protein with Both UGM and UNGM be other basic amino acids, histidine and lysine, respectively, in
Activity—This study represents the first demonstration of an the E. coli and K. pneumoniae UGM. In M. tuberculosis, Arg-59
enzyme involved in the interconversion of UDP-GalpNAc and was also replaced by histidine; however, Arg-168 was found to
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 499

Bifunctional Pyranose-Furanose Mutase from C. jejuni
venting the formation of nonpro-
ductive oxazoline-like intermedi-
ates, which could be formed by an
intramolecular reaction of the acet-
amido group (Fig. 8). When the sub-
strate is UDP-Gal, then there is no
possibility of forming such interme-
diates. Therefore, Arg-59 does not
aid in catalysis. Instead, because
arginine is more bulky than histi-
dine, it could lower the catalytic rate
due to steric interactions in the
active site during catalysis.
The mutagenesis of both R59H
and R168K results in a larger
decrease in turnover of UDP-
GalfNAc substrate while simulta-
neously increasing turnover of
UDP-Galf. This results in an
increased selectivity for UDP-Galf
than observed for either of the sin-
gle mutants (Fig. 7; Table 1). Some-
FIGURE 8. Formation of oxazoline intermediate. A, acetamido group of GalNAc can undergo an unproduc-
tive intramolecular reaction to form an FAD-bound oxazoline intermediate that prevents conversion to
GalfNAc. B, possible interaction between Arg-59 and the GalNAc acetamido group that would prevent forma-
tion of an oxazoline intermediate and promote conversion to GalfNAc.
be a threonine rather than the lysine observed in E. coli and K. what surprisingly, the increase in the Km value for UDP-
pneumoniae.
GalfNAc seen for the R168K mutant was not observed in the
Mutagenesis of R168K only resulted in a 2-fold increase in case of the double mutant. This suggests that the amino acids
selectivity for UDP-Galf over UDP-GalfNAc in the co-incuba- play a synergistic role in allowing the enzyme to interconvert
tion assay; however, there were more significant changes UDP-GalfNAc and UDP-GalpNAc, rather than an additive role
observed for the kinetic parameters. This amino acid change in which Arg-59 is the major determinant.
resulted in a decrease in the catalytic activity of the protein for
Subtle Amino Acid Substitutions Result in Changes in C.
both substrates compared with the wild-type UNGM, but most jejuni UNGM Substrate Tolerance—Despite the high sequence
interesting was that this substitution resulted in a significant identity of C. jejuni UNGM with the E. coli, M. tuberculosis, and
increase in the Km value with UDP-GalfNAc but not with UDP- K. pneumoniae UGM, the UNGM possesses the ability to rec-
Galf. As the Km value approximates the dissociation constant ognize UDP-GalNAc as a substrate. Most of the sequence var-
for the enzyme-substrate complex, this supports the notion iability occurs in solvent-exposed residues, and the amino acids
that Arg-168 has a role in binding and stabilizing UDP-GalNAc making up the active site are nearly identical in all four mutase
in the active site. It has previously been proposed that amino enzymes (supplemental Fig. S2, boxed residues). The two resi-
acid residues in the mobile loop of UGMs are involved in sub- dues identified here as playing an important role in allowing
strate recognition (19, 20). As Arg-168 is located in the mobile recognition of UDP-GalNAc are relatively conservative re-
loop, its ability to stabilize the UDP-GalNAc enzyme-substrate placements of the residues found in the other mutase enzymes;
complex is consistent with the role of the mobile loop in sub- however, they nevertheless have a significant effect on the sub-
strate recognition. This arginine residue is located 4.2 Å from strate selectivity of the enzyme. It should be appreciated that
the acetamido carbonyl oxygen in our homology model with small variations in amino acids leading to changes in substrate
docked UDP-GalpNAc (Fig. 6A), indicating a possible hydro- specificity are well known in carbohydrate-active enzymes. For
gen bonding interaction.
example, the blood group GTA and GTB glycosyltransferases,
Mutation of the other active site arginine (Arg-59) indicates which use UDP-GalpNAc versus UDP-Galp, respectively, as the
that it also plays an important role in the catalytic activity of donor substrate, differ in only four amino acids (61). Similarly,
UNGM. Mutagenesis of R59H results in a greater than 4-fold in Neisseria meningitidis, a single amino acid change in the
decrease in kcat for UDP-GalfNAc, while simultaneously result- capsule polymerase determines the substrate specificity for
ing in a 3-fold increase in kcat for UDP-Galf. The Km value is also either Glcp or Galp transferase activity (62).
changed for both substrates, but this appears minor in compar-
Although the two arginine residues identified in this study influ-
ison with the observed changes in kcat. Considering our homol- ence the substrate specificity, the mutagenesis of either residue or
ogy model, Arg-59 is located within 4.5 Å of the carbonyl oxy- both failed to result in a complete loss in UNGM activity. It is
gen of the acetamido group of UDP-GalpNAc (Fig. 6A). In the therefore clear that other amino acids further removed from the
E. coli UGM crystal structure, the equivalent residue His-59 sits active site also contribute to the specificity of the enzyme, and
below the active site (data not shown) and does not protrude these remain to be elucidated. As well, we have demonstrated that
into the active site as does Arg-59 in UNGM. Therefore, it C. jejuni UNGM can function as a UGM in vivo; however, there
appears that Arg-59 is able to interact with the UDP-GalNAc havebeennoGalfresiduesreportedinC. jejuniglycoconjugates. In
substrate to stabilize the intermediate. This may occur by pre- this context, another unresolved issue is if other C. jejuni strains
500 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 1•JANUARY 1, 2010

Bifunctional Pyranose-Furanose Mutase from C. jejuni
Senchenkova, S. N., Knirel, Y. A., and Savage, A. V. (1999) Carbohydr. Res.
319, 124–132
containing the glf gene possess Galf-containing glycoconjugates,
and investigations into the specificity of the transferases accepting
donors from bifunctional enzymes are warranted. This study fur-
ther demonstrates the intricacies in bacterial glycoconjugate bio-
synthesis. Detailed understanding of these systems will allow for
the development of novel antimicrobials targeting these patho-
gen-specific pathways.
32. Arbatsky, N. P., Shashkov, A. S., Mamyan, S. S., Knirel, Y. A., Zych, K., and
Sidorczyk, Z. (1998) Carbohydr. Res. 310, 85–90
33. St Michael, F., Szymanski, C. M., Li, J., Chan, K. H., Khieu, N. H., Larocque,
S., Wakarchuk, W. W., Brisson, J. R., and Monteiro, M. A. (2002) Eur.
J. Biochem. 269, 5119–5136
34. Eppe, G., Peltier, P., Daniellou, R., Nugier-Chauvin, C., Ferrie`res, V., and
Vincent, S. P. (2009) Bioorg. Med. Chem. Lett. 19, 814–816
35. Wang, Q., Ding, P., Perepelov, A. V., Xu, Y., Wang, Y., Knirel, Y. A., Wang,
L., and Feng, L. (2008) Mol. Microbiol. 70, 1358–1367
REFERENCES
1. Peltier, P., Euzen, R., Daniellou, R., Nugier-Chauvin, C., and Ferrie`res, V.
(2008) Carbohydr. Res. 343, 1897–1923
36. Allos, B. M. (2001) Clin. Infect. Dis. 32, 1201–1206
2. Richards, M. R., and Lowary, T. L. (2009) ChemBioChem 10, 1920–1938
3. de Lederkremer, R. M., and Colli, W. (1995) Glycobiology 5, 547–552
4. Whitfield, C. (1995) Trends Microbiol. 3, 178–185
37. Kaldor, J., and Speed, B. R. (1984) Br. Med. J. 288, 1867–1870
38. Delederkremer, R. M., Nahmad, V. B., and Varela, O. (1994) J. Org. Chem.
59, 690–692
5. Brennan, P. J., and Nikaido, H. (1995) Annu. Rev. Biochem. 64, 29–63
6. Lee, R. E., Smith, M. D., Nash, R. J., Griffiths, R. C., McNeil, M., Grewal,
R. K., Yan, W. X., Besra, G. S., Brennan, P. J., and Fleet, G. W. (1997)
Tetrahedron Lett. 38, 6733–6736
39. Errey, J. C., Mukhopadhyay, B., Kartha, K. P., and Field, R. A. (2004) Chem.
Commun. 2706–2707
40. Miller, W. G., Bates, A. H., Horn, S. T., Brandl, M. T., Wachtel, M. R., and
Mandrell, R. E. (2000) Appl. Environ. Microbiol. 66, 5426–5436
41. Marolda, C. L., Welsh, J., Dafoe, L., and Valvano, M. A. (1990) J. Bacteriol.
172, 3590–3599
7. Pan, F., Jackson, M., Ma, Y., and McNeil, M. (2001) J. Bacteriol. 183,
3991–3998
8. Pedersen, L. L., and Turco, S. J. (2003) Cell. Mol. Life Sci. 60, 259–266
9. Bela´nova´, M., Dianiskova´, P., Brennan, P. J., Completo, G. C., Rose, N. L.,
Lowary, T. L., and Mikusova´, K. (2008) J. Bacteriol. 190, 1141–1145
10. Nassau, P. M., Martin, S. L., Brown, R. E., Weston, A., Monsey, D., McNeil,
M. R., and Duncan, K. (1996) J. Bacteriol. 178, 1047–1052
11. Ko¨plin, R., Brisson, J. R., and Whitfield, C. (1997) J. Biol. Chem. 272,
4121–4128
42. Carrillo, C. D., Taboada, E., Nash, J. H., Lanthier, P., Kelly, J., Lau, P. C.,
Verhulp, R., Mykytczuk, O., Sy, J., Findlay, W. A., Amoako, K., Gomis, S.,
Willson, P., Austin, J. W., Potter, A., Babiuk, L., Allan, B., and Szymanski,
C. M. (2004) J. Biol. Chem. 279, 20327–20338
43. Yao, R., Alm, R. A., Trust, T. J., and Guerry, P. (1993) Gene 130, 127–130
44. Nothaft, H., Liu, X., McNally, D. J., Li, J., and Szymanski, C. M. (2009) Proc.
Natl. Acad. Sci. U.S.A. 106, 15019–15024
12. Weston, A., Stern, R. J., Lee, R. E., Nassau, P. M., Monsey, D., Martin, S. L.,
Scherman, M. S., Besra, G. S., Duncan, K., and McNeil, M. R. (1997) Tuber
Lung Dis. 78, 123–131
45. Bacon, D. J., Szymanski, C. M., Burr, D. H., Silver, R. P., Alm, R. A., and
Guerry, P. (2001) Mol. Microbiol. 40, 769–777
46. Tsai, C. M., and Frasch, C. E. (1982) Anal. Biochem. 119, 115–119
47. Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J.,
and Valvano, M. A. (1999) J. Biol. Chem. 274, 35129–35138
48. Marolda, C. L., Lahiry, P., Vine´s, E., Saldías, S., and Valvano, M. A. (2006)
Methods Mol. Biol. 347, 237–252
13. Beverley, S. M., Owens, K. L., Showalter, M., Griffith, C. L., Doering, T. L.,
Jones, V. C., and McNeil, M. R. (2005) Eukaryotic Cell 4, 1147–1154
14. Bakker, H., Kleczka, B., Gerardy-Schahn, R., and Routier, F. H. (2005) Biol.
Chem. 386, 657–661
15. Sanders, D. A., Staines, A. G., McMahon, S. A., McNeil, M. R., Whitfield,
C., and Naismith, J. H. (2001) Nat. Struct. Biol. 8, 858–863
16. Zhang, Q., and Liu, H. (2000) J. Am. Chem. Soc. 122, 9065–9070
17. Soltero-Higgin, M., Carlson, E. E., Gruber, T. D., and Kiessling, L. L. (2004)
Nat. Struct. Mol. Biol. 11, 539–543
49. Lambert, C., Le´onard, N., De Bolle, X., and Depiereux, E. (2002) Bioinfor-
matics 18, 1250–1256
50. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew,
R. K., and Olson, A. J. (1998) J. Comput. Chem. 19, 1639–1662
51. Bian, X. L., Rosas-Acosta, G., Wu, Y. C., and Wilson, V. G. (2007) J. Virol.
81, 2899–2908
18. Beis, K., Srikannathasan, V., Liu, H., Fullerton, S. W., Bamford, V. A.,
Sanders, D. A., Whitfield, C., McNeil, M. R., and Naismith, J. H. (2005) J.
Mol. Biol. 348, 971–982
52. Fersht, A. (1999) in Structure and Mechanism in Protein Science: A Guide
to Enzyme Catalysis and Protein Folding (Julet, M. R., ed) pp. 116–117,
W. H. Freeman & Co., New York
19. Yao, X. H., Bleile, D. W., Yuan, Y., Chao, J., Sarathy, K. P., Sanders, D. A.,
Pinto, B. M., and O’Neill, M. A. (2009) Proteins Struct. Funct. Bioinformat.
74, 972–979
53. Parkhill, J., Wren, B. W., Mungall, K., Ketley, J. M., Churcher, C., Basham,
D., Chillingworth, T., Davies, R. M., Feltwell, T., Holroyd, S., Jagels, K.,
Karlyshev, A. V., Moule, S., Pallen, M. J., Penn, C. W., Quail, M. A., Rajan-
dream, M. A., Rutherford, K. M., van Vliet, A. H., Whitehead, S., and
Barrell, B. G. (2000) Nature 403, 665–668
20. Chad, J. M., Sarathy, K. P., Gruber, T. D., Addala, E., Kiessling, L. L., and
Sanders, D. A. R. (2007) Biochemistry 46, 6723–6732
21. Caravano, A., Sinay¨, P., and Vincent, S. P. (2006) Bioorg. Med. Chem. Lett.
16, 1123–1125
54. Liu, D., and Reeves, P. R. (1994) Microbiology 140, 49–57
55. Stevenson, G., Neal, B., Liu, D., Hobbs, M., Packer, N. H., Batley, M., Red-
mond, J. W., Lindquist, L., and Reeves, P. (1994) J. Bacteriol. 176, 4144–4156
56. Lee, R., Monsey, D., Weston, A., Duncan, K., Rithner, C., and McNeil, M.
(1996) Anal. Biochem. 242, 1–7
22. Itoh, K., Huang, Z., and Liu, H. W. (2007) Org. Lett. 9, 879–882
23. Carlson, E. E., May, J. F., and Kiessling, L. L. (2006) Chem. Biol. 13, 825–837
24. Zhang, Q., and Liu, H. (2001) J. Am. Chem. Soc. 123, 6756–6766
25. Yuan, Y., Bleile, D. W., Wen, X., Sanders, D. A., Itoh, K., Liu, H. W., and
Pinto, B. M. (2008) J. Am. Chem. Soc. 130, 3157–3168
57. Karlyshev, A. V., Champion, O. L., Churcher, C., Brisson, J. R., Jarrell,
H. C., Gilbert, M., Brochu, D., St Michael, F., Li, J., Wakarchuk, W. W.,
Goodhead, I., Sanders, M., Stevens, K., White, B., Parkhill, J., Wren, B. W.,
and Szymanski, C. M. (2005) Mol. Microbiol. 55, 90–103
58. Guan, S., Clarke, A. J., and Whitfield, C. (2001) J. Bacteriol. 183, 3318–3327
59. Bernatchez, S., Szymanski, C. M., Ishiyama, N., Li, J., Jarrell, H. C., Lau,
P. C., Berghuis, A. M., Young, N. M., and Wakarchuk, W. W. (2005) J. Biol.
Chem. 280, 4792–4802
26. Errey, J. C., Mann, M. C., Fairhurst, S. A., Hill, L., McNeil, M. R., Naismith,
J. H., Percy, J. M., Whitfield, C., and Field, R. A. (2009) Org. Biomol. Chem.
7, 1009–1016
27. Yuan, Y., Wen, X., Sanders, D. A., and Pinto, B. M. (2005) Biochemistry 44,
14080–14089
28. Gruber, T. D., Borrok, M. J., Westler, W. M., Forest, K. T., and Kiessling,
L. L. (2009) J. Mol. Biol. 391, 327–340
29. Gruber, T. D., Westler, W. M., Kiessling, L. L., and Forest, K. T. (2009)
Biochemistry 48, 9171–9173
60. Cha, S. (1968) Mol. Pharmacol. 4, 621–629
61. Rose, N. L., Palcic, M. M., and Evans, S. V. (2005) J. Chem. Educ. 82,
1846–1852
30. Feng, L., Senchenkova, S. N., Yang, J., Shashkov, A. S., Tao, J., Guo, H.,
Cheng, J., Ren, Y., Knirel, Y. A., Reeves, P. R., and Wang, L. (2004) J.
Bacteriol. 186, 4510–4519
62. Claus, H., Stummeyer, K., Batzilla, J., Mu¨hlenhoff, M., and Vogel, U. (2009)
Mol. Microbiol. 71, 960–971
31. Hanniffy, O. M., Shashkov, A. S., Moran, A. P., Prendergast, M. M.,
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 501

Glycobiology and Extracellular Matrices:
Characterization of a Bifunctional
Pyranose-Furanose Mutase from
Campylobacter jejuni 11168
Myles B. Poulin, Harald Nothaft, Isabelle
Hug, Mario F. Feldman, Christine M.
Szymanski and Todd L. Lowary
J. Biol. Chem. 2010, 285:493-501.
doi: 10.1074/jbc.M109.072157 originally published online November 3, 2009
Access the most updated version of this article at doi: 10.1074/jbc.M109.072157
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2009/11/03/M109.072157.DC1.html
This article cites 60 references, 20 of which can be accessed free at
http://www.jbc.org/content/285/1/493.full.html#ref-list-1