(Received for publication, September 16, 1996, and in revised form, November 11, 1996)
From the Canadian Bacterial Diseases Network,
Department of Microbiology, University of Guelph,
Guelph, Ontario, Canada, N1G 2W1, and the § Institute
for Biological Sciences, National Research Council, Ottawa,
Ontario, Canada, K1A 0R6
The O-side-chain polysaccharide in the
lipopolysaccharide of Klebsiella pneumoniae O1 is based on
a backbone structure of repeat units of
[3)-
-D-Galf-(1
3)-
-D-Galp-(1
];
this structure is termed D-galactan I. The rfb
(O-antigen biosynthesis) gene cluster directs the synthesis of
D-galactan I and consists of six genes termed
rfbA-FKPO1. In this paper we show that
rfbDKPO1 encodes a UDP-galactopyranose mutase
(NAD(P)H-requiring) (EC 5.4.99.9), which forms uridine 5
-(trihydrogen
diphosphate) P
-
-D-galactofuranosyl ester
(UDP-Galf), the biosynthetic precursor of galactofuranosyl residues. The deduced amino acid sequence of
rfbDKPO1 shows 85% and 37.5% identity to the
rfbDKPO8 gene of K. pneumoniae
serotype O8 and the glf gene of Escherichia
coli, respectively. The molecular mass of the purified
RfbDKPO1 enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophoresis, while gel filtration revealed a
molecular mass of 92 kDa, suggesting a dimeric structure for the native
protein. The rfbDKPO1 gene product
interconverts uridine 5
-(trihydrogen diphosphate)
P
-
-D-galactopyranosyl ester (UDP-Galp) and
UDP-Galf. Unlike Glf, RfbDKPO1 showed a
requirement for NADH or NADPH, which could not be replaced by NAD or
NADP. RfbDKPO1 was used to synthesize milligram quantities
of UDP-Galf, allowing this compound to be purified and
fully characterized in an intact form for the first time. The structure
of UDP-Galf was proven by NMR spectroscopy.
Lipopolysaccharide (LPS)1 is a major
component of the outer membrane of Gram-negative bacteria. In enteric
bacteria, the LPS molecule comprises a hydrophobic lipid A portion,
which forms the outer leaflet of the outer membrane, a core
oligosaccharide, and an O-side-chain polysaccharide. The O
polysaccharide varies in structure from strain to strain, giving rise
to unique antigenic epitopes (O antigens). In the genus
Klebsiella, there exists a family of structurally related
galactose-containing O polysaccharides. These are based on a backbone
structure consisting of a disaccharide O-repeat unit
[3)-
-D-Galf-(1
3)-
-D-Galp-(1
]
known as D-galactan I (1). Variations in O antigens arise
from addition of side-chain
-D-Galp (2) and
O-acetyl (2, 3) residues, or by addition of domains of
varying structure (1, 4-6). Galactofuranosyl residues are present in a
growing number of LPS O antigens e.g. in strains of
Serratia marcescens (7, 8), Shigella dysinteriae (9), Shigella boydii (10), Escherichia coli (11),
Pasteurella hemolytica (12), Hemophilus
pleuropneumoniae (13), and Actinobacillus pleuropneumoniae (14). The T1-antigen polysaccharide of
Salmonella friedenau (15) and a variety of capsular or
extracellular polysaccharides from both Gram-negative and Gram-positive
bacteria (e.g. Refs. 16-20) contain Galf.
Galactofuranosyl residues are a central component in the
mycolyl-arabinogalactan complex, which is characteristic of the cell
walls of mycobacteria (21) and the related genera Nocardia
and Rhodococcus (22), in the lipoglycan of Mycoplasma mycoides (23), in the paracrystalline S-layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 (24), and in the
cellulosome glycoproteins of Clostridium thermocellum (25)
and Bacteroides cellulosolvens (26). In eukaryotes,
galactofuranosyl residues are found in the lipophosphoglycan of
Leishmania donovani (27), Leishmania major (28),
and Leishmania mexicana (29), in the N-linked
glycoproteins of Crithidia spp. (30), and in the
lipopeptidophosphoglycan of Trypanosoma cruzi (31). A
variety of fungal cell surface glycans, glycolipids and glycoproteins
contain Galf residues in Penicillium spp.
(32-34), Aspergillus spp. (35), Neurospora
crassa (36), and Histoplasma capsulatum (37). Many of
these microorganisms are important pathogens, and, for some, current
therapies are limited. This observation, together with the absence of
Galf residues in human glycoconjugates has fueled interest
in the potential of generating novel therapeutic compounds directed
against reactions involved in the formation of Galf
precursors (38).
In the prototype system in K. pneumoniae O1,
D-galactan I biosynthesis is directed by enzymes encoded by
six genes in the chromosomal rfb locus. The polymer is
synthesized in the cytoplasm by RfbCDEF activities and is then
transported across the plasma membrane by a process involving an
ATP-binding cassette (ABC-2) transporter, where RfbA is the
transmembrane component and RfbB contains the consensus ATP-binding
motifs (39). The synthesis of D-galactan I is initiated on
a "primer" comprising undecaprenyl pyrophosphoryl
N-acetylglucosamine. The primer is formed by the activity of
the Rfe protein which appears to be an UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase (40). RfbF is a bifunctional galactosyl
transferase, which forms the disaccharide
[3)-
-D-Galf-(1
3)-
-D-Galp-1(
] on the primer (41). The details of the subsequent polymerization reaction(s) have not yet been resolved.
The precursor for Galp residues is UDP-Galp (42), formed by the action of UDP-galactose 4-epimerase, encoded by the galE gene in the galactose operon (43). Early studies on the biosynthesis of the T1 antigen in S. enterica serovar Typhimurium (44, 45) suggested that Galf precursors were derived from Galp at the level of UDP-linked sugars. The Galf precursor for a galactofuranosyl-containing polysaccharide in Penicillium charlesii was also proposed to be a UDP-linked derivative (46). More recently, the product of orf6, an enzyme encoded by the cryptic rfb locus in E. coli K-12 strains (47), was shown to have UDP-galactopyranose mutase (EC 5.4.99.9) activity, capable of the reversible formation of UDP-Galf from UDP-Galp (48). The product UDP-Galf was predicted from degradation products and this compound has not been isolated and fully characterized in an intact state.
This paper reports the cloning, expression, and purification of RfbDKPO1 from K. pneumoniae O1. We show that the enzyme has UDP-galactopyranose mutase activity but differs in several important aspects from the homologue from E. coli K-12. In addition, we report the isolation and first complete chemical characterization of intact UDP-Galf.
The strains and plasmids used in this study are given in Table I. Strains were grown in Luria-Bertani (LB) medium containing the appropriate antibiotics. Antibiotics were used at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; chloramphenicol, 50 µg/ml.
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Restriction endonuclease digestion, ligation, and transformation were performed essentially as described by Sambrook et al. (49). Plasmid DNA preparations were made with QIAGEN Inc. columns according to the manufacturer's instructions. CLUSTAL W was used for the multiple alignment (50) using default parameters.
Plasmid ConstructionpWQ71 was constructed by cloning a
XbaI-HindIII fragment of pWQ5 containing the
complete rfbKPO1 gene cluster into pBBR1MCS. In
order to delete an internal region in rfbDKPO1,
plasmid pWQ5 was mutagenized using a Transformer site-directed
mutagenesis kit (Clontech), which is based on the method of Deng and
Nickoloff (51). The 5-phosphorylated primers 5
-CAT GTT TAT
CAT ATT TTC C-3
and 5
-GAT TAC CAG
GCA GTG ATG-3
were used to introduce a
BamHI site (underlined) in the 5
and 3
-regions of
rfbDKPO1, respectively. The 5
-phosphorylated
selection primer 5
-GCC TTT TTA CTT AGT AAT CCT AAA G-3
alters a
unique CelII site in rfbFKPO1 and was
used as selection primer. Digestion of this construct with
BamHI, followed by religation, resulted in an in-frame
deletion of 630 base pairs. The rfbKPO1 gene
cluster containing the rfbDKPO1 deletion was
subsequently cloned into pBBR1MCS on a
XbaI-HindIII fragment, resulting in pWQ70. To
clone the rfbDKP01 open reading frame, plasmid
pWQ5 was mutagenized using the Transformer site-directed mutagenesis
kit (Clontech) in order to introduce NdeI and
HindIII sites overlapping with the start and stop codons of
rfbDKPO1, respectively. The
NdeI-HindIII fragment containing the entire
rfbDKPO1 open reading frame was cloned into
pET30a(+) (Novagen), resulting in pWQ66.
LPS samples were routinely prepared by the SDS-proteinase K lysate method (52). Samples were analyzed by SDS-PAGE according to the conditions described by Lesse et al. (53), and gels were silver-stained by the method of Tsai and Frasch (54).
Overexpression of RfbDKPO1An overnight culture
of E. coli CWG288 (pWQ66) was diluted 1:200 into fresh
medium (LB, 250 mM sorbitol, 50 µg/ml kanamycin) and
grown to an A580 of 0.5. Galactose and
isopropyl-1-thio--D-galactopyranoside were added to a
final concentration of 10 mM and 0.4 mM,
respectively. The cells were then grown for an additional 3 h at
37 °C, collected by centrifugation at 7,800 × g,
4 °C for 10 min, and stored at
70 °C in aliquots of 1 g of
wet cell paste.
For the extraction of sugar nucleotides, 1 g of wet cell paste was resuspended in 5 ml of 75% ethanol and incubated at 100 °C for 10 min. Cell debris was removed by centrifugation (20,000 × g, 10 min) and reextracted with another 5 ml of 75% ethanol. The samples were evaporated to dryness and redissolved in a small volume of water. Insoluble material was removed by centrifugation.
Purification of the RfbDKPO1 Protein of K. pneumoniae Serotype O1Wet cell paste (1 g) was resuspended in 9 ml of 50 mM HEPES, pH 7.0, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, and the suspension was disrupted by sonication. Cell debris was removed by centrifugation at 20,000 × g for 30 min, followed by ultracentrifugation at 100,000 × g for 1 h. The 100,000 × g supernatant was further fractionated by ammonium sulfate precipitation at 4 °C. The fraction between 55% and 75% saturation contained the RfbDKPO1 protein and was dialyzed against 50 mM HEPES pH 7.0 overnight, and then concentrated to ~ 0.5 ml using a ultrafiltration cartridge (Centriplus 30, Amicon). RfbD was further purified by dye-ligand affinity chromatography on 5-ml columns of Reactive Green 5-agarose (Sigma) and Cibacron Blue 3GA-agarose (Sigma) at room temperature. The concentrated protein fraction was applied to the Reactive Green 5-column preequilibrated with 50 mM HEPES pH 7.0 and washed with 25 ml of 50 mM HEPES pH 7.0. This column bound the majority of applied protein, while most of the RfbDKPO1 protein was not retained under this conditions. The unbound fraction was subsequently applied to a 5-ml column of Cibacron Blue 3GA-agarose equilibrated with 50 mM HEPES pH 7.0, washed with 25 ml of 50 mM HEPES pH 7.0. The rfbDKP01 protein was eluted with 10 ml of 1 mM UDP-Galp in 50 mM HEPES pH 7.0. Residual contaminating proteins were removed by anion exchange chromatography on MonoQ at room temperature, using a linear sodium chloride gradient (0-300 mM) in 50 mM HEPES pH 7.0.
Enzyme ReactionsUDP-galactopyranose mutase reactions were performed at 37 °C for 30 min. The reaction mixture consisted in a total volume of 100 µl: 50 mM HEPES pH 7.0, 1 mM UDP-Galp (UDP-Galf), 2 mM NADH or NADPH, and variable amounts of UDP-galactopyranose mutase (NAD(P)H-requiring). The reaction products were analyzed by HPAEC on CarboPac PA1 (Dionex).
HPLC AnalysisSugar nucleotides were analyzed on a CarboPac PA1 column (4 × 250 mm) according to the method of Palmieri et al. (55) with minor modifications. Sugar nucleotides were separated using a linear ammonium acetate (pH 7.0) gradient from 200 to 500 mM in 50 min at a flow rate of 0.6 ml/min. UDP-sugars were detected at 262 nm. For the purification of larger quantities of UDP-Galf, a CarboPac PA1 column (9 × 250 mm) operated at a flow rate of 3 ml/min was used. Separated compounds were recovered by lyophilization.
NMR AnalysisThe 1H, 13C, and 31P spectra were recorded on a Bruker AMX-600 spectrometer, at 285 K using a 5-mm broadband tunable probe. The sample was prepared from 0.5 mg of UDP-Galf in ammonium acetate buffer, lyophilized, and redissolved in 0.6 ml of 10 mM potassium phosphate buffer (pH 7.2), containing 0.5 mM EDTA. The pH of the sample dropped below 6; therefore, additional K2HPO4 was immediately added to adjust the pH to 6.8. The sample was then lyophilized and redissolved in 0.6 ml of D2O and transferred to a 5-mm NMR tube. Acetone was added to the sample to provide the internal proton chemical shift reference at 2.225 ppm. The methyl resonance of an external acetone in D2O, set at 31.07 ppm, was used for the 13C chemical shift reference. The chemical shift reference for 31P was that of external phosphoric acid (25%) in D2O set at 0.0 ppm. All of the experiments were carried out without sample spinning and with the standard software and pulse programs provided by Bruker. The J(H,H) and J(P,H) coupling constants were measured directly from the one-dimensional 1H or 31P spectra processed with a digital resolution of 0.2 Hz/point.
The 1H spectrum of 256 scans was recorded with presaturation of the HOD resonance at 4.945 ppm. The 13C spectrum with proton decoupling was recorded overnight with 50,000 scans. The 31P spectrum of 4000 scans was recorded without proton decoupling. The two-dimensional homonuclear magnitude COSY, phase-sensitive TOCSY, 1H-31P HMQC, 1H-13C HMQC experiments were recorded and processed as described previously (56). The one-dimensional TOCSY z-filtered spectra of 2000 scans was performed as described previously (57).
The
rfbKPO1 gene cluster that directs formation of
D-galactan I (39, 42) consists of six genes termed
rfbA-F. The rfbDKPO1 open reading
frame comprises 1152 nucleotides. No consensus ribosome binding site
was found upstream of the ATG initiation codon of rfbDKPO1. The TGA stop codon of
rfbDKPO1 overlaps with the ATG initiation codon
of rfbEKPO1, suggesting translational coupling. The predicted translational product of rfbDKPO1
is a 384-amino acid protein with a molecular weight of 44,454 and a
theoretical pI of 6.06. The N-terminal region between Lys-5 and Asp-33
contains a signature for an ADP-binding -fold involved in FAD
or NAD binding (58). The sequence deviates only at Gly-19 from the fingerprint. Consequently, a BLAST search (59) revealed striking similarities of the N terminus of rfbDKPO1 to
flavin-containing oxidases and dehydrogenases. Across the entire
predicted polypeptide, 85% and 37.5% identity were found to the
rfbDKPO8 gene of K. pneumoniae serotype O8 (also comprising D-galactan I (3, 60) and the glf (orf6) gene of E. coli K-12, respectively
(Fig. 1).
Direct evidence that rfbDKPO1 encodes a Glf
homologue was obtained from complementation experiments. The
rfbKPO1 gene cluster was cloned into pBBR1MCS
resulting in pWQ71. Site-directed mutagenesis was used to generate
plasmid pWQ70, which contains an in-frame deletion of 630 base pairs in
the rfbDKPO1 gene of the D-galactan I gene cluster. A physical map of the DNA fragments cloned into pWQ70
or pWQ71 is given in Fig. 2. The LPS phenotypes
conferred by these plasmids were analyzed by SDS-PAGE in DH5 and a
derivative of strain SØ874 (CWG287). The glf gene, located
in the cryptic E. coli K-12 rfb region (47, 61),
is deleted in E. coli SØ874. The LPS of DH5
or CWG287
both containing the complete cluster (pWQ71) showed a ladder of smooth
LPS (S-LPS) (Fig. 3). Deletion of the internal region of
rfbDKPO1 in pWQ70 resulted in no phenotypic alterations in DH5
, while in CWG287 the synthesis of S-LPS was completely abolished (Fig. 3), suggesting that a gene of the cryptic rfb cluster of E. coli K-12, most likely
glf, is functionally equivalent to rfbD
KPO1.
An E. coli K-12
The function of RfbDKPO1 was further
addressed by analyzing the UDP-sugars synthesized in vivo.
In order to achieve high level expression of RfbDKPO1, a
NdeI and a HindIII site overlapping with the
start and stop codon of rfbDKPO1, respectively,
were introduced by site-directed mutagenesis. The
NdeI-HindIII fragment containing only the
rfbDKPO1 open reading frame was cloned into the
T7 expression vector pET30a(+), resulting in pWQ66. The functionality of the rfbDKPO1 gene was proven by
complementation of CWG287 (pWQ70). When pWQ66 was transformed into
CWG287 (pWQ70), the synthesis of S-LPS was restored, even without
isopropyl-1-thio--D-galactopyranoside induction (Fig.
3). To avoid further metabolism of in vivo synthesized UDP-Galp or UDP-Galf, CWG288 (E. coli
K-12
rfb
DE3 galE) was constructed as a
galE derivative of CWG287. Additionally, sorbitol, which has
been reported to suppress elevated levels of UDP-sugar hydrolase
activity as a result of UDP-galactose accumulation (62), was added to
the medium to avoid enzymatic degradation. Nucleotide sugars were
extracted from CWG288 (pWQ66) and from the control strain CWG288
(pET30a(+)), and extracts were analyzed by HPAEC. From Fig.
4 it can be seen that both strains accumulated
UDP-Galp (retention time 34.5 min), while an additional peak
(p37) with a retention time of 37.5 min was unique to extracts of
CWG288 (pWQ66). In experiments using [1-14C]galactose,
p37 was found to contain about 5% of the radioactivity, with the
remaining 95% confined to the UDP-Galp peak (data not shown), suggesting that p37 is a novel galactose-containing sugar nucleotide formed by the activity of RfbDKPO1.
Purification and Characterization of the RfbDKPO1 Protein
In order to unequivocally determine the function of
RfbDKPO1 and the nature of p37, the RfbDKPO1
protein was purified to homogeneity from CWG288 (pWQ66) by ammonium
sulfate precipitation, dye-ligand affinity chromatography, and anion
exchange chromatography as described in detail under "Experimental
Procedures." The protein was homogeneous as judged by SDS-PAGE (Fig.
5). The molecular mass of 45,000 Da of the denatured
polypeptide determined by SDS-PAGE is in good agreement with the
predicted molecular weight of 44,454. However, gel filtration on
Superose 12 revealed a molecular weight of 92,000, suggesting a dimeric
structure for the native protein (data not shown).
An in vitro assay was used to gain more information on the
RfbDKPO1-mediated reaction. The enzyme reaction was carried
out in a total volume of 100 µl, and the reaction products obtained after incubation at 37 °C for 30 min were analyzed by HPAEC on a
CarboPac PA1. The enzyme showed an absolute requirement for NADH or
NADPH (data not shown). Under these reaction conditions, a peak well
separated from UDP-Galp or UDP-Glc with the same retention time as p37 was obtained (Fig. 6B). In all
experiments about 5% of UDP-Galp was converted to p37. No
reaction could be seen in assays lacking the cofactor, or in assays
containing NAD or NADP. When pooled p37 was used as substrate in the
enzyme assay, ~95% was converted back to UDP-Galp (Fig.
6C), while no reaction was observed in assays lacking a
cofactor or in assays with NAD or NADP (data not shown). Other than the
cofactor requirements, these data are in agreement with the data
described for Glf of E. coli (48) and further support the
conclusion that p37 indeed is UDP-Galf.
NMR Analysis of UDP-Galf
In order to unequivocally prove that
p37 is UDP-Galf, about 1.5 mg of p37 was pooled from several
HPLC runs on a semipreparative CarboPac PA1 (9 × 250 mm). After
lyophilization, a small amount was redissolved in 100 mM
ammonium acetate, pH 7.0, and analyzed by HPAEC. The material was found
to be >95% pure (Fig. 6D) as judged by peak integration.
During preparation of the sample for NMR analysis, the pH of the sample
dropped below pH 6.0, which resulted in partial degradation of the
nucleotide sugar. However, a complete assignment by two-dimensional
methods of all proton, carbon, and phosphorous NMR resonances permitted
the identification of intact p37 and of all the major components of the
sample. By integration of the 1H spectrum, the sample was
found to be a mixture of the following major components: 26%
UDP-Galf, 53% 5-UMP, and 21% Galf-1,2-P. The
1H spectrum is shown in Fig. 7, together
with subspectra for the Galf moiety of UDP-Galf
and Galf-1,2-P. The 1H-13C HMQC is
shown in Fig. 8A along with the
1H and 13C spectra. The
1H-31P HMQC spectrum is shown in Fig.
8B, along with the 1H and 31P
spectra. Proton, 13C and 31P chemical shifts
are given in Tables
II, III, IV. Coupling constants for 31P are in Table V and
J(H,H) coupling constants in Table
VI. Due to limited quantities of material, the low
signal to noise in the 13C spectrum did not permit the
measurement of J(C,P) couplings.
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The 31P spectrum showed the presence of
various phosphorylated compounds, one of which contained a
pyrophosphate group that has characteristic shifts at 11.2 ppm and
12.4 ppm and a J(P
,P
) coupling constant
of 21 Hz (63). A 1H-31P HMQC spectrum indicated
the proton resonances, which were coupled to the pyrophosphate group.
The proton resonances at 5.629 ppm and 4.152 ppm, which were coupled to
the 31P resonance at
12.4 ppm had
J(P,H) coupling constants of 5.5 Hz and 2.3 Hz,
respectively. The complete coupled spin system for these proton
resonances was identified by COSY, TOCSY, and one-dimensional TOCSY
experiments. The proton-proton coupling constants
(J(H,H)) could be obtained from the
one-dimensional TOCSY spectrum. A 1H-13C HMQC
permitted the assignment of all the carbon atoms directly bonded to
these protons (C-H). The proton chemical shifts,
J(H,H) coupling constants, 13C
chemical shifts (64), and 31P chemical shifts were all
characteristic of a terminal
-Galf residue bonded to a
pyrophosphate group.
From a similar analysis, the resonances at 4.24 and 4.21 ppm, which
bonded to the pyrophosphate resonance at 11.2 ppm, were found to
belong the H5
and H5" resonances of the ribose moiety of
UDP-Galf. The proton chemical shifts,
J(H,H) coupling constants, and
J(P,H) coupling constants of the UDP moiety were
found to be characteristic for those found for UDP sugars (65).
In the 1H-31P HMQC spectrum,
the 31P resonances at 3.5 ppm bonded to the proton
resonances at 4.032 ppm and 3.970 ppm were found to belong to 5-UMP by
a complete assignment of the 1H and 13C
spectra. The proton chemical shifts, J(H,H)
coupling constants, and J(P,H) coupling
constants of the 5
-UMP were found to be similar to those previously
reported for this compound (66).
The 31P resonance at 17.1 ppm is characteristic of a five-membered cyclic ester (63). In the 1H-31P HMQC spectrum, this 31P resonance was coupled to the proton resonances at 6.000 ppm and 4.813 ppm, with J(P,H) coupling constants of 14 Hz and 6.8 Hz, respectively. From the assignment of the proton spin system for this residue obtained from the COSY and one-dimensional TOCSY experiment, the H1 and H2 resonances were found to be coupled to the cyclic phosphate.
Galactofuranosyl residues are found in a growing number of surface
glycoconjugates from both Gram-negative and Gram-positive bacteria, as
well as protozoa and fungi. These include well documented pathogens of
humans and livestock. In bacteria (this work, and Refs. 44, 45, and 48)
and fungi (46), the biosynthetic precursor for Galf residues
is believed to be UDP-Galf. Galactofuranosyl-containing glycoconjugates have not been reported in humans so far, which makes
UDP-Galf formation an interesting target for novel
therapeutic compounds (38). However, development of strategies for
inhibitors has been limited by the lack of fundamental information
regarding the biosynthesis of galactofuranosyl residues.
Galactocarolose, a -D-(1
5)-linked
polygalactofuranosid produced by P. charlesii, was the first
polysaccharide found to contain galactofuranosyl residues (34, 67).
Trejo et al. (46) isolated a nucleotide sugar formed in
cell-free enzyme preparations of P. charlesii, which was
capable of acting as a donor of galactofuranosyl residues in the
biosynthesis of a galactofuranosyl-containing polymer. Based on
chemical analysis of the reaction products obtained by acid and
alkaline hydrolosis as well as periodate oxidation, Trejo et
al. (46) predicted that the sugar nucleotide is
UDP-Galf. More recently, Nassau et al. (48)
cloned the glf gene of E. coli and showed that it
encodes UDP-galactopyranose mutase (EC 5.4.99.9). Nassau et
al. (48) concluded that the reaction product of the
UDP-galactopyranose mutase reaction is UDP-Galf based on
HPLC analysis of the sugar 1-phosphate obtained after phosphodiesterase
treatment of the reaction product. The modification of an HPAEC
procedure, to give a method that separates UDP-Galp from
UDP-Galf, allowed us for the first time to purify milligram quantities of UDP-Galf and to unequivocally prove by NMR the
structure to be uridine 5
-(trihydrogen diphosphate)
P
-
-D-galactofuranosyl ester. This will provide an
essential reagent for biochemical analyses of synthetic systems for
galactofuranosyl-containing glycoconjugates.
The data presented here establish that RfbDKPO1 catalyzes
the interconversion of UDP-Galp and UDP-Galf. The
LPS profiles of E. coli K-12 strains containing pWQ70 and
pWQ71 clearly demonstrated that Glf (48) of E. coli can
complement the rfbDKPO1 deletion in pWQ70. Glf
and RfbDKPO1 are therefore functionally equivalent. Although the K. pneumoniae RfbDKPO1 protein
shows 37.5% identity to Glf of E. coli, the two proteins do
exhibit remarkable differences. Glf has been reported to be relatively
unstable, resulting in complete loss of activity after storage of the
purified protein at 4 °C for >24 h (48). In contrast,
RfbDKPO1 can be stored at 4 °C for periods in excess of
1-2 weeks without significant loss of activity (data not shown).
Moreover, no cofactor requirement other than FAD has been reported for
Glf, while RfbDKPO1 has an absolute requirement for NADH or
NADPH. These differences are difficult to explain. One possibility is
that the binding constants for NADH (NADPH) are different in both
proteins. Alternatively, the differences may be a result of the
purification procedure used. Cibacron Blue 3GA used in the purification
procedure reported here is thought to bind to NAD binding sites.
Consequently, binding of the enzyme to this resin may displace the
essential cofactor. Analysis of the predicted sequence of both proteins
revealed a ADP-binding -fold at its N terminus, which could be
involved in binding FAD or NAD. The motif in K. pneumoniae
differs at Gly-19 from the fingerprint. However, the known structure of
p-hydroxybenzoate hydroxylase, which has a glycine at the
same position (position 15 of the fingerprint), demonstrates that
peptides with a glycine at this position can form a functional
unit (68). The presence of FAD in the purified Glf protein
has been reported (48). Although we have not analyzed this cofactor in
RfbDKPO1, the yellow color of the pure protein is
consistent with its containing FAD. Gel filtration of the
RfbDKPO1 protein on Superose 12 revealed a molecular weight
of 92,000 for the native protein. The possible dimerization of Glf has
not been investigated.
Little is known about the mechanism of the UDP-galactopyranose mutase (NAD(P)H-requiring) reaction. Stevenson et al. (47) suggested that the reaction may proceed via a 2-keto intermediate. Since in our hands the UDP-galactopyranose mutase reaction shows an absolute requirement for NADH or NADPH and was found to be inactive with NAD or NADP, an alternative is that the first step of the reaction may be a reduction step, although there is no net oxidation/reduction in the interconversion of UDP-Galp and UDP-Galf. The stability of the RfbDKPO1 protein and the possibility to synthesize and purify larger quantities of UDP-Galf will facilitate a detailed analysis of the reaction mechanism and determination of the protein structure.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L31762[GenBank] (for rfbCDEFKPO1).
We gratefully acknowledge the technical assistance provided by Karen Sutherland. We thank K. Duncan and colleagues for discussions and sharing data on glf prior to its publication.
While this manuscript was in press, a report by others independently confirmed the nature and structure of UDP-Galf as the product of the UDP-galactopyranose mutase reaction (Lee, R., Monsey, D., Weston, A., Duncan, K., Rithner, C., and McNeil, M. (1996) Anal. Biochem. 242, 1-7).