Identification of a bifunctional UDP-4-keto-pentose/UDP-xylose synthase in the plant pathogenic bacterium Ralstonia solanacearum strain GMI1000, a distinct member of the 4,6-dehydratase and decarboxylase family

Article abstract of DOI:10.1074/jbc.M109.066803

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-β-L-threo-pentopyranosyl- 4″-ulose in the presence of NAD⁺. The second activity converts UDP-β-L-threo-pentopyranosyl-4″-ulose and NADH to UDP-xylose and NAD⁺, albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.

Full text of DOI:10.1074/jbc.M109.066803

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 12, pp. 9030–9040, March 19, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose
Synthase in the Plant Pathogenic Bacterium Ralstonia
solanacearum Strain GMI1000, a Distinct Member of
□
S
the 4,6-Dehydratase and Decarboxylase Family*
Received for publication, September 16, 2009, and in revised form, January 15, 2010 Published, JBC Papers in Press, January 29, 2010, DOI 10.1074/jbc.M109.066803
Xiaogang Gu‡, John Glushka‡, Yanbin Yin§, Ying Xu§, Timothy Denny¶, James Smith‡, Yingnan Jiang‡,
and Maor Bar-Peled‡ʈ1
From the ‡Complex Carbohydrate Research Center, the §Computational System Biology Laboratory, Department of Biochemistry
and Molecular Biology, and the Institute of Bioinformatics, and the Departments of ¶Plant Pathogen and ʈPlant Biology, University
of Georgia, Athens, Georgia 30602
The UDP-sugar interconverting enzymes involved in UDP-
GlcA metabolism are well described in eukaryotes but less is
known in prokaryotes. Here we identify and characterize a gene
(RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which
encodes a dual function enzyme not previously described. One
activity is to decarboxylate UDP-glucuronic acid to UDP-␤-L-
merly Pseudomonas aeruginosa) (10, 11); in the gum-like
polysaccharides secreted by Aeromonas nichidenii (12); and
recently in the O-antigenic polysaccharide of the Gram-nega-
tive plant endosymbiont Rhizobium leguminosarum (13). How
genes encoding enzymes forming UDP-xylose were evolved
and why xylosyl residue appears predominantly in eukaryotes
but scarce in bacteria remains unclear.
؉
threo-pentopyranosyl-4ꢀ-ulose in the presence of NAD . The
second activity converts UDP-␤-L-threo-pentopyranosyl-4ꢀ-ulose
The metabolism of UDP-glucuronic acid (UDP-GlcA) varies
among species (Fig. 1A). In animals, UDP-GlcA is reported to
be converted only to UDP-xylose by UDP-GlcA decarboxylase
(also named UDP-xylose synthase (Uxs)) (14), whereas plants
can interconvert UDP-GlcA to UDP-apiose by UDP-apiose
synthase (also named UDP-apiose/UDP-xylose synthase
(Uaxs)) (15, 16); to UDP-galacturonic acid (UDP-GalA) by
UDP-GlcA 4-epimerase (17); and also to UDP-xylose by plant
Uxs (18). Uaxs also produces UDP-xylose in vitro, albeit at a lower
ratio compared with UDP-apiose (16). In addition, one bacterium
wasreportedtoconvertUDP-GlcAtoUDP-L-Ara4Obytheaction
of C-terminal ArnA as an intermediate for the synthesis of UDP-
␤-4-deoxy-4-formamido-L-arabinose (19).
؉
and NADH to UDP-xylose and NAD , albeit at a lower rate. Our
dataalsosuggestthatfollowingdecarboxylation, thereisstereospe-
cific protonation at the C5 pro-R position. The identification of the
R. solanacearum enzyme enables us to propose that the ancestral
enzymeof UDP-xylosesynthase andUDP-apiose/UDP-xylose syn-
thase was diverged to two distinct enzymatic activities in early bac-
teria. This separation gave rise to the current UDP-xylose synthase
in animal, fungus, and plant as well as to the plant Uaxs and bacte-
rial ArnA and U4kpxs homologs.
UDP-xylose is an essential metabolite required to initiate
synthesis of proteoglycans in humans and animals (1–3), syn-
thesis of the acidic polysaccharide glucuronoxylomannan in the
pathogenic fungus Cryptococcus neoformans (4–6), and syn-
thesis of diverse plant polysaccharides such as xylan, xyloglu-
can, and xylogalacturonan (7, 8). Interestingly, xylosyl residues
are rarely found in the bacteria or archaea analyzed to date.
However, trace amounts of xylose were reported to be present
in the lipopolysaccharide (LPS)2 of certain strains of the human
pathogens Proteus mirabilis (9) and Ralstonia aeruginosa (for-
We are interested in studying the metabolism of UDP-GlcA
across all species with an aim to understand the origin and role of
glycan diversity in biology. Such information may explain how the
addition of new glycosyl residues to the specific polysaccharides
provides new functions. Here, we report the first identification of a
gene in the plant pathogen Gram-negative bacterium Ralstonia
solanacearum str. GMI1000, which encodes an enzyme capable of
converting UDP-GlcA to UDP-4-keto-pentose and subsequently
at lower rate to UDP-xylose (Fig. 1B). The identification of this
enzyme provides new insights related to UDP-GlcA metabolism
and may help to explain how early plants or other eukaryotes
obtained Uxs or Uaxs genes.
* This work was supported by National Science Foundation Grant IOB-
0453664 (to M. B.-P.) and in part by the BioEnergy Science Center, which is
supported by the Office of Biological and Environmental Research in the
Department of Energy Office of Science.
EXPERIMENTAL PROCEDURES
□S
Phylogeny Analysis of Plant Uxs and Uaxs Homologs—The
BLAST program (20) was used to identify proteins in the NCBI
nonredundant data base that share high sequence similarity to
functional plant Uaxs. Similarly, protein sequences of known
Uxs were used to identify homologous proteins with an E value
range between 1e-110 and 3e-25. The Uxs-like and Uaxs-like
protein sequence data sets were merged. The CD-HIT program
was used to select proteins where any two would have sequence
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Tables S1 and S2 and Figs. S1–S4.
1 To whom correspondence should be addressed: CCRC 315 Riverbend Rd.,
Athens, GA 30602. Fax: 706-542-4412; E-mail: peled@ccrc.uga.edu.
2 The abbreviations used are: LPS, lipopolysaccharide; UDP-4-keto-pentose,
UDP-␤-L-threo-pentopyranosyl-4Љ-ulose; RsU4kpxs, R. solanacearum UDP-
4-keto-pentose/UDP-xylose synthase; UDP-GlcA, UDP-glucuronic acid;
Uxs, UDP-xylose synthase; Uaxs, UDP-apiose/UDP-xylose synthase; HPLC,
high pressure liquid chromatography; MES, 4-morpholineethanesulfonic
acid; L-Ara4N, 4-amino-4-deoxy-L-arabinose.
9030 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 12•MARCH 19, 2010

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
FIGURE 1. UDP-GlcA metabolism in eukaryote and prokaryote. A, in animals, some fungi and plants, UDP-GlcA is converted to UDP-xylose by UDP-GlcA
decarboxylase (also named Uxs). Plants and several bacteria (such as Streptococcus pneumoniae) consist of UDP-GlcA 4-epimerase (UGlcAE) that interconverts
UDP-GlcA and UDP-GalA. Plants also have a bifunctional UDP-apiose synthase/Uaxs that can interconvert UDP-GlcA to UDP-apiose and UDP-xylose. In some
bacteria, UDP-GlcA can be converted into UDP-L-Ara4O in the presence of ArnA. B, the proposed enzymatic activities of UDP-4-keto-pentose synthase and
subsequent UDP-xylose synthase. In the presence of NADϩ, UDP-GlcA is decarboxylated to UDP-4-keto-pentose and NADH. The final protonation at C5 is
stereoselective (HR in bold type). In solution, UDP-4-keto-pentose exists predominantly as the gem-diol hydrate form (see bottom right). The U4kpxs enzyme is
bifunctional, and the released UDP-4-keto-pentose in the presence of NADH can be converted to UDP-xylose and NADϩ, albeit at a lower rate when compared
with the first activity. HR and HS indicate the pro-R H5 and pro-S H5 of UDP-4-keto-pentose, whereas Ha and He indicate the axial H5 and equatorial H5 of
UDP-xylose, respectively.
identity lower than 95%. The processed sequence data were was used to perform maximum likelihood phylogeny recon-
reduced to a final set of total 135 proteins. The conserved Pfam struction using the PhyML v2.4.4 program (22). Several protein
epimerase domains in the final set proteins were used for the candidates were selected for further work.
multiple sequence alignment and for the subsequent phyloge-
cDNA Cloning—Genomic DNA was isolated from R.
netic analysis. The multiple sequence alignment was built using solanacearum str. GMI1000. The coding sequence
the MAFFT v6.603 program (21), and the resulting alignment (NP_519440.1) of the putative Ralstonia protein that has an E
MARCH 19, 2010•VOLUME 285•NUMBER 12
JOURNAL OF BIOLOGICAL CHEMISTRY 9031

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
value of 4e-62 to the potato Uaxs (StUaxs) (16) and an E value of GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 7.6, con-
9e-32 to Arabidopsis Uxs3 (AtUxs3) (18) was amplified by PCR taining 0.1 M NaCl. The eluant was monitored at 280 nm, and
using 1 unit of high fidelity proofreading Platinum DNA poly- the fractions were collected every 30 s. The fractions containing
merase (Invitrogen) and 0.2 ␮M of each forward and reverse enzyme activity were pooled and kept at Ϫ80 °C.
primers: 5Ј-TCATGAAGAAAGTACTGATCCTCGGCGTC-3Ј
HPLC-based Enzyme Assay—Unless otherwise indicated,
and 5Ј-AAGCTTGTCGACCAGGCTGCGCGCC-3Ј. The PCR RsU4kpxs reactions (final volume, 50 ␮l) consisted of 50 mM
ϩ
product was cloned to generate plasmid pGEM-T:RsGM#4 and sodium phosphate, pH 7.6, 1 mM NAD , 1 mM UDP-GlcA, and
verified by sequencing (GenBankTM accession number 1 ␮g of recombinant RsU4kpxs. The reactions were kept at
GQ369438). The BsphI-HindIII fragment (1061bp) containing 37 °C for up to 20 min and then terminated by boiling (100 °C, 2
the full-length gene (from now on named UDP-4-keto-pen- min). An equal volume of chloroform was added, and after vor-
tose/UDP-xylose synthase (RsU4kpxs)) without the stop codon texing (30 s) and centrifugation (12,000 rpm for 5 min, at room
was subcloned into an Escherichia coli expression vector temperature), the upper aqueous phase was collected and chro-
derived from pET28b (23) to generate pET28b:RsU4kpxs#1 matographed (17) on a Q15 anion exchange column (2-mm
that will add six histidines to the C terminus of the recombinant inner diameter ϫ 250 mm long; Amersham Biosciences) using
RsU4kpxs.
an Agilent Series 1100 HPLC system equipped with an
Protein Expression and Purification—E. coli cells containing autosampler, diode array detector and ChemStation software.
pET28b:RsU4kpxs#1 or an empty vector (control) were cul- Alternatively, reaction products were separated on a Prep C18
tured for 16 h at 37 °C in LB medium (20 ml) supplemented with Scalar column (4.6-mm inner diameter ϫ 250 mm long; Agi-
kanamycin (50 ␮g/ml) and chloramphenicol (34 ␮g/ml). A por- lent) using 20 mM tert-butylamine-H3PO4, pH 6.6, in 20%
tion (7 ml) of the cultured cells was transferred into fresh LB liquid methanol. The nucleotides were detected by their UV absor-
medium (250 ml) supplemented with the same antibiotics, and the bance using the Agilent photodiode array detector. The maxi-
ϩ
cells were then grown at 37 °C at 250 rpm until the cell density mum absorbance for UDP-sugars, NAD , and NADH were
reached A600 ϭ 0.6. The cultures were then transferred to 30 °C, 261, 259, and 259/340 nm, respectively, in ammonium formate
and gene expression was induced by the addition of isopropyl ␤-D- and tert-butylamine-H3PO4, pH 6.6, in 20% methanol. The
thiogalactoside to a final concentration of 0.5 mM. After 4 h of peak areas of analytes were compared with calibration curves
growth while shaking (250 rpm), the cells were harvested by cen- of internal standards (UDP-Glc, UDP-GlcA, and NADH)
trifugation (6,000 ϫ g for 10 min at 4 °C), resuspended in lysis and used to calculate the amount of products formed. The
buffer (10 ml 50 mM sodium phosphate, pH 7.5, containing 10% enzyme reaction was also done in a D2O solution as
(v/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phen- described above, except that the buffer was made in 99%
ylmethylsulfonylfluoride), andlysedinanicebathby24sonication D2O, to which the enzyme was added, giving a final solution
cycles (10-s pulse; 20-s rest) using a Misonix S-4000 (Misonix Inc., containing 90% D2O.
1
Farmingdale, NY) equipped with microtip probe. The lysed cells
Real Time H NMR-based Assay—Two types of real time
were centrifuged at 4 °C for 10 min at 6,000 ϫ g, and the superna- NMR assays were performed at 37 °C each in a total volume of
tant was supplemented with 1 mM dithiothreitol and centrifuged 180 ␮l. The D2O reactions were performed in a final mixture of
again (30 min at 20,000 ϫ g). The resulting supernatant (termed D2O:H2O (9:1 v/v) and contained 50 mM sodium phosphate,
ϩ
s20) was recovered and kept at Ϫ20 °C.
pH, pD 7.6, 1 mM UDP-GlcA, 1 mM NAD , and 3 ␮g of recom-
His-tagged protein was purified on a column (10-mm inner binant RsU4kpxs. The purified enzyme was supplied in H2O
diameter ϫ 150 mm long) containing 2 ml of nickel-Sepharose buffer. The H2O reactions were identical but performed in a
(Qiagen) equilibrated with 50 mM sodium phosphate, pH 7.5, con- final solution of 10% D2O and 90% H2O. After the addition of
taining 0.3 M NaCl. The bound His-tagged protein was eluted with the enzyme, the reaction mixture was transferred to a 3-mm
the same buffer containing increasing concentrations of imid- NMR tube. Real time 1H NMR spectra were obtained using a
azole. The fractions containing RsU4kpxs activity were pooled, Varian DirectDriveTM 600-MHz spectrometer equipped
supplemented with 1 mM dithiothreitol, flash frozen in liquid with a cryogenic probe. Data acquisition was started ϳ5 min
nitrogen, and stored in aliquots at Ϫ80 °C. Proteins extracted from after the addition of enzyme to optimize the spectrometer.
E. coli cells expressing empty vector were passed via the same Sequential one-dimensional proton spectra with presatura-
nickel column, and fractions eluted with imidazole were collected tion of the water resonance were acquired over the course of
and served as controls in enzyme assays and SDS-PAGE analyses. the enzymatic reaction. Spectra of individual 1 mM standards
ϩ
The concentration of proteins was determined using the Bradford of NAD , UDP-xylose, UDP-GlcA, and NADH prepared in
reagent using bovine serum albumin as standard.
the same buffer were collected with the same temperature
The molecular masses of the recombinant proteins were esti- and parameter settings. Additional NMR analyses were per-
mated by size exclusion chromatography using a Waters 626 formed using Varian DirectDriveTM 900-MHz spectrome-
LC HPLC system equipped with a photodiode array detector ter. All of the spectra at 37 °C were referenced to the water
(PDA 996) and a Waters Millennium32 work station. Separate resonance at 4.63 ppm downfield of 2,2-dimethyl-2-silapen-
solutions (0.5 ml) of RsU4kpxs or a mixture of standard pro- tane-5-sulfonate; the spectra at 25 °C were referenced to
teins (2 mg each of aldolase (157 kDa), bovine serum albumin water at 4.76 ppm.
(68 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4
Enzyme Properties of RsU4kpxs—The enzyme activity was
kDa)) were separately chromatographed at 0.5 ml minϪ1 on a tested in a variety of buffers, at different temperatures, different
Superdex75 column (10-mm inner diameter ϫ 300 mm long; ions, or with different potential inhibitors. For optimal pH
9032 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 12•MARCH 19, 2010

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
FIGURE 2. Protein sequence alignments and phylogenetic relationship of Uxs-like and Uaxs-like proteins from diverse species. A, phylogenetic rela-
tionships of selected Uxs-like and Uaxs-like proteins. Protein sequences were aligned and analyzed using the MAFFT program, and the phylogenetic trees were
created using the PhyML program. The bootstrap values for the clades are shown. The bar represents 0.5-amino acid substitutions/site. B, protein sequences
of Arabidopsis AtUxs3 (NP_001078768, amino acids 40–375), of C. neoformans Uxs1p (AAM22494, amino acids 83–410), of potato StUaxs (ABC75032, amino
acids 7–386), of Arabidopsis AtAxs1(AAC73015, amino acids 10–389), of ArnA (AY057445, amino acids 311–660), of Erwinia tasmaniensis ArnA homolog,
Et-ArnA (YP_001908305, amino acids 311–660), and of full-length RsU4kpxs (GQ369438) were aligned with ClustalX software, and the conserved motifs are
shown.
experiments, 1 ␮g of recombinant enzyme was first mixed with and the amount of UDP-4-keto-pentose was measured by
1 mM UDP-GlcA and 50 mM of each individual buffer (Tris- HPLC.
ϩ
HCl, sodium phosphate, MES, or HEPES). NAD (1 mM) was
RESULTS
then added, and after 20 min of incubation at 37 °C, the amount
of UDP-4-keto-pentose formed was determined by HPLC.
Identification, Cloning, and Characterization of Ralstonia UDP-
Inhibition assays were performed by first mixing the enzyme ␤-L-threo-pentopyranosyl-4Љ-ulose Synthase, RsU4kpxs—As de-
and sodium phosphate buffer with various additives (e.g. scribed in the introduction, several enzymes are known to
ϩ
nucleotides) on ice for 10 min. UDP-GlcA and NAD (1 mM interconvert UDP-GlcA to other UDP-sugars. These proteins
each) were then added. After 20 min at 37 °C, the amount of contain two conserved motifs that are also common in other
UDP-4-keto-pentose formed was calculated from the HPLC NDP-sugar interconverting enzymes such as 4,6-dehydratases,
chromatogram. For the optimal temperature experiments, and 4-epimerases (24). At the N-terminal region of these pro-
ϩ
the assays were performed under standard conditions except teins is the GXXGXXG domain that binds the cofactor NAD
that reactions were incubated at different temperature for 20 (25), and ϳ100 amino acids downstream is the YXXXK motif
min. Subsequently, the activities were terminated (100 °C), (26) that is involved in catalysis. Aside from these conserved
MARCH 19, 2010•VOLUME 285•NUMBER 12
JOURNAL OF BIOLOGICAL CHEMISTRY 9033

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
motifs, the above enzymes do have amino acid differences that
account for their substrate specificity and catalytic properties.
The maximum likelihood phylogenetic tree shown in Fig. 2A
indicates that there are five major clades for proteins related to
Uxs and Uaxs: a eukaryote Uxs clade, a plant Uaxs clade, an
ArnA (19) clade, and two additional clades (A and B) with
unknown functions. Members belonging to clades A, B, and
ArnA are mostly bacteria (Fig. 2A). A clade B protein from R.
solanacearum str. GMI1000 was chosen as a possible link to the
ancestral origin of enzymes that produce UDP-apiose and
UDP-xylose. There are other bacterial proteins in clade B. Some
belong to human pathogenic species such as Burkholderia
cenocepacia, but others are nonpathogenic species including
Geobacter bemidjiensis. The R. solanacearum protein that we
selected shares 28% amino acid identity to Arabidopsis AtUxs3,
35% identity to potato StUaxs, and higher identity (53%) to the
bacterial ArnA C-terminal domain from E. coli (Fig. 2B). The
Ralstonia protein differs from ArnA having a short six-amino
acid extension peptide after the GXXGXXG motif and appears
to share no sequence similarity in amino acids 270–290 and
330–350 (supplemental Fig. S1). To determine whether these
amino acid differences alter the specific activity of the Ralstonia
enzyme, the encoded gene was expressed in E. coli, purified,
and subsequently characterized.
Compared with control, a highly expressed protein band (41
kDa) was detected after SDS-PAGE analysis of E. coli cells
expressing recombinant R. solanacearum protein (Fig. 3A, lane
1). The protein was column-purified (Fig. 3A, lane 3), and pre-
liminary HPLC-based experiments demonstrated that both the
FIGURE 3. Expression and initial characterization of recombinant
RsU4kpxs. A, SDS-PAGE of total soluble protein isolated from E. coli cells
expressing RsU4kpxs (lane 1) or control empty vector (lane 2), and after puri-
ficationovernickelcolumnLane3,RsU4kpxs;lane4,control.B,HPLCchromato-
gram of RsU4kpxs enzyme reaction. Purified recombinant RsU4kpxs was
incubated with UDP-GlcA for 40 min in the presence (panel 2) or absence
(panel 4) of exogenous NADϩ. As a control, the corresponding column-puri-
fied protein isolated from cells expressing control empty vector was incu-
bated with UDP-GlcA and NADϩ for 40 min (panel 3). The reaction products
were separated with a Q15 column. The distinct UDP-sugar peak (marked by
arrow, in panel 2) was collected and analyzed by NMR, and the NADH peak in
panel 2 is marked by an asterisk.
ϩ
crude and purified protein converts UDP-GlcA and NAD to a
UDP-sugar and NADH. NADH was confirmed as an enzymatic
product based on its specific dual UV absorbance at 259 and
340 nm, and its HPLC chromatograph was identical as authen-
tic NADH standard (Fig. 3B, panel 2). To determine the identity
of the other product, the reaction with purified enzyme was
separated by Q15 anion exchange column, and the product
peak eluted at 17.2 min (Fig. 3B, panel 2, marked by arrow) was
FIGURE 4. 1H NMR analysis of the RsU4kpxs product, UDP-␤-L-threo-pentopyranosyl-4ꢀ-ulose (UDP-4-keto-pentose). The enzymatic product (Fig. 3B,
panel 2, marked by arrow) was collected and analyzed by NMR at 37 °C. One-dimensional 600-MHz NMR spectrum of the product is shown in selected regions
between 3.5 and 4.4 and between 5.5 and 8.0 ppm. The location for each proton residue on the spectrum is indicated: the position of protons on the uracil (Ura)
ring are indicated by H, the ribose (Rib) protons are indicated by HЈ, and the 4-keto-pentose protons are indicated by HЉ. Note the diagnostic doublet signal for
each of C5 protons: pro-R H5 (3.51ppm) and pro-S H5 (3.89 ppm).
9034 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 12•MARCH 19, 2010

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
TABLE 1
Enzymatic kinetics of the bi-functional recombinant RsU4kpxs
Synthase activity
UDP-4-keto-pentosea
Km, UDP-GlcA (␮M)
22.2 Ϯ 1.2
113.2 Ϯ 4.1
4.2 Ϯ 0.3
ϩ
Km, NAD (␮M)
Ϫ1
V
max, UDP-GlcA (␮M min
)
Ϫ1
ϩ
Vmax, NAD (␮M min
)
4.7 Ϯ 0.3
Ϫ1
k
cat, UDP-GlcA (min
)
17.1 Ϯ 1.2
19.2 Ϯ 1.4
12.8 Ϯ 0.3
2.8 Ϯ 0.3
Ϫ1
ϩ
kcat, NAD (min
)
Ϫ1
k
k
cat/Km (UDP-GlcA) (mMϪ1 s
)
cat/Km (NAD ) (mMϪ1 s
)
ϩ Ϫ1
UDP-xyloseb
K
K
V
V
m, UDP-4-keto-pentose (␮M)
m, NADH (␮M)
max, UDP-4-keto-pentose (␮M min
152.9 Ϯ 22.3
94.2 Ϯ 12.7
3.6 Ϯ 0.2
Ϫ1
)
Ϫ1
max, NADH (␮M min
)
3.9 Ϯ 0.1
Ϫ1
k
k
k
k
cat, UDP-4-keto-pentose (min
)
0.73 Ϯ 0.05
0.81 Ϯ 0.01
0.081 Ϯ 0.009
0.15 Ϯ 0.01
Ϫ1
cat, NADH (min
)
cat/Km (UDP-4-keto-pentose) (mMϪ1 s
)
Ϫ1
cat/Km (NADH) (mMϪ1 s
)
Ϫ1
a UDP-4-keto-pentose synthase activity was measured with varied concentrations of
ϩ
UDP-GlcA (0.08–1.0 mM) or NAD (0.08–1.0 mM) in the presence of 0.5 ␮g of
enzyme after 10 min at standard conditions. The reciprocal initial velocity was
ϩ
plotted against the reciprocal UDP-GlcA or NAD concentration according to
Lineweaver and Burk to calculate the corresponding Km values. The data pre-
sented are the average Km values from three experiments.
b UDP-xylose synthase activity was measured with varied concentrations of UDP-4-
ϩ
keto-pentose (0.075–0.3 mM) or NAD (0.1–0.4 mM) in the presence of 10 ␮g of
enzyme after 20 min of reaction. The reciprocal initial velocity was plotted against
the reciprocal UDP-4-keto-pentose or NADH concentration according to Lin-
eweaver and Burk to calculate the corresponding Km values. The data presented
are the average Km values from three experiments.
mum activity at 37 °C (Fig. 5B). RsU4kpxs was inhibited by UDP
and NADH but not by other nucleotides or nucleotide-sugars
(supplemental Table S3). To investigate cofactor and nucleo-
tide-sugar specificity, the enzyme was reacted for up to 60 min
FIGURE5. Theeffectsofbuffercomposition,pHandtemperatureonUDP-
4-keto-pentose synthase activity. A, the activity of recombinant RsU4kpxs
wasanalyzedatdifferentbuffers(Tris-HCl, sodiumphosphate, MES, orHEPES)
at different pH. Each value is the mean of triplicate reactions. B, the activity of
recombinant RsU4kpxs was analyzed at different temperatures in 50 mM
sodium phosphate buffer, pH 7.6. Each value is the mean of triplicate
reactions.
ϩ
ϩ
with the NAD analog NADP or with the UDP-GlcA isomer
UDP-GalA, and in both cases activity was not observed (data
not shown). Thus we concluded that the recombinant enzyme
ϩ
is specific for NAD and UDP-GlcA. The molecular size of the
active RsU4kpxs was determined by size exclusion chromatog-
raphy. The active fraction migrated as a globular protein having
collected and analyzed by 1H NMR. The NMR spectrum (Fig. 4 a mass between 80 and 120 kDa, suggesting that RsU4kpxs is
and supplemental Table S1) provided chemical shifts consis- not a monomer, possibly a dimer. Kinetics analyses of the UDP-
tent with UDP-␤-L-threo-pentopyranosyl-4Љ-ulose (herein 4-keto-pentose synthase activity of RsU4kpxs are summarized
referred to as UDP-4-keto-pentose), previously reported as the in Table 1. The apparent Km values were 22.2 (UDP-GlcA) and
ϩ
enzymatic product of ArnA (19). Consistent with the report by 113.2 ␮M (NAD ), Vmax values (␮M minϪ1) were 4.2 for UDP-
ϩ
Ϫ1
Ϫ1
Breazeale et al. (19), no nuclear Overhauser effect was observed GlcA and 4.7 for NAD , and the kcat/Km values (s mM
)
ϩ
between the H5 protons and any other ring protons (data not were 12.8 (UDP-GlcA) and 2.8 (NAD ).
shown), preventing the identification of pro-R and pro-S H5 by
Real Time NMR Analysis of UDP-4-keto-pentose Synthase,
conventional methods. However, the NMR analysis of selec- RsU4kpxs—1H NMR spectroscopy is an important tool to mon-
tively deuterated enzymatic products discussed later in this itor enzymatic reactions in real time (16, 17, 27, 28). This pro-
paper provided the assignment of H5 protons that is reversed cedure is ideal for analyzing the time-dependent appearance
from the Breazeale et al. (19) report. Although the compound and disappearance of intermediates and the detection of unsta-
has been shown to exist primarily in the gem-diol hydrated ble products.
form in aqueous solution (19), it is depicted in Fig. 4 as the
NMR-based RsU4kpxs enzyme reactions were first done in
keto-form for simplicity. Thus the function of the gene encod- 90% D2O to reduce the interference of the water signal. Over
ing RsU4kpxs protein is confirmed as a UDP-4-keto-pentose the time of the reaction, as shown in Fig. 6A, the peaks corre-
ϩ
synthase.
sponding to NAD (N) and UDP-GlcA (G) decreased, and
Characterization and Properties of Recombinant UDP-4- simultaneously, peaks corresponding to NADH (H) and UDP-
keto-pentose Synthase—The purified recombinant enzyme is 4-keto-pentose (K) increased. However, in addition to the
stable if it was flash frozen after purification. Like Uxs and Uaxs, expected proton doublet for K5s (3.89 ppm), an additional sin-
RsU4kpxs does not require metals for activity and remains fully glet peak (marked by an arrow in Fig. 6A, and see inset for an
active in the presence of EDTA (supplemental Table S2). The expansion view) was also observed to increase at the same rate.
enzyme is active between pH 5 and pH 9 (Fig. 5A) with maxi- To identify the molecule that gives rise to the singlet peak, the
MARCH 19, 2010•VOLUME 285•NUMBER 12
JOURNAL OF BIOLOGICAL CHEMISTRY 9035

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
FIGURE 6. Real time 1H NMR analyses of UDP-4-keto-pentose synthase activity performed in D2O. A, purified recombinant RsU4kpxs was mixed with
UDP-GlcA, buffer, and NADϩ (in a final mixture of 9:1 D2O/H2O). Approximately 5 min after enzyme addition and NMR shimming, NMR data were collected (bottom
trace). Selectedregions(from2.6to4.2, 5.5to6.1, and7.0to9.4ppm)ofthespectrafrom5to35minareshown. Thesignalsarelabeledasindicatedbythecompounds
above the spectrum: protons belonging to NADϩ (N), NADH (H), UDP-4-keto-pentose (K), and UDP-GlcA (G). The single peak (indicated by an arrow) for UDP-4-keto-
pentosepro-SH5isduetotheselectivedeuterationofitspro-RH5.Theinsetshowsexpansionofthepro-SH5signal(s)thatarosefromamixtureofdeuteratedandfully
protonated molecular species of UDP-4-keto-pentose. The enzyme activity during real time NMR was carried out at 37 °C. Peaks marked by * (from 7.9 to 8.0 ppm) are
the mixture of signals for Ura-H6 proton of UDP-4-keto-pentose and UDP-GlcA. Peaks marked by ** (from 3.5 to 3.7ppm) are the mixture of signals for the G2, K2, K5R,
and glycerol protons derived from the enzyme preparation. Peaks marked by *** (from 5.9 to 6.0 ppm) are the mixture of signals for Rib-H1Ј and Ura-H5 protons of
UDP-4-keto-pentoseandUDP-GlcA. Xindicatesanimpurityresonance. B, recombinantRsU4kpxswasincubatedwithNADϩ, UDP-GlcA, andbufferinafinalmixtureof
90% D2O. The enzymatic product was separated by HPLC, and the UDP-4-keto-pentose peak was collected and analyzed by 1H NMR. One-dimensional 600-MHz NMR
spectrum (operated at 37 °C) of the UDP-4-keto-pentose is shown from 3.5 to 4.5 and 5.5 to 8.0 ppm. The signals are labeled as in Fig. 3, and the single peak for
UDP-4-keto-pentosepro-SH5,wherepro-RH5isdeuterated,isindicatedbyanarrow.Integrationofpeaksshowsthat60%areindeuteratedUDP-4-keto-pentoseform
(D) and 40% are protonated (H).
9036 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 12•MARCH 19, 2010

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
FIGURE 8. Real time 1H NMR analyses of UDP-4-keto-pentose synthase
activity performed in H2O. Purified recombinant RsU4kpxs was mixed with
UDP-GlcA, buffer, and NADϩ in a final mixture of H2O/D2O (9:1 v/v). Approx-
imately 5 min after enzyme addition and NMR shimming, the NMR data were
collected (bottom trace). The enzyme activity during real time NMR was car-
ried out at 37 °C. Selected region of the 1H NMR spectra from 2.6 to 4.2 ppm
and from 5 to 35 min are shown. The signals are labeled as indicated by the
compounds shown in Fig. 6A. The doublet peak of the pro-S H5 of UDP-4-
keto-pentose is marked by an arrow. The peaks marked by ** (from 3.5 to
FIGURE 7. Illustration of NMR spectra for enzymatic derived UDP-4-keto-
3.7ppm) are mixtures of signals for the G2, K2, K5R, and glycerol protons
derived from the enzyme preparation.
pentose species. A, UDP-4-keto-pentose is made as fully protonated (panel I,
C5-HR/HS) and pro-R H5 deuterated (panel II, C5-DR/HS) forms. B, simulated
NMR spectrum when both C5 HS and HR are protonated (panel 1), and when
C5 pro-R proton is deuterated (DR) (panel 2). Note the pro-S in panel 2 is pro-
tonated (HS). Panel 3 shows the simulated spectrum of the mixture (panels I mechanism of Williams et al. (29) and Gatzeva-Topalova et al.
and II). The pro-R and pro-S protons at C5 are labeled as H5R and H5S, respec-
tively. The pro-S proton where the pro-R position is deuterated is labeled as
(30), who proposed that following the abstraction of the C4
hydride and formation of the UDP-4-keto sugar, decarboxyla-
H5S(d). The simulations (Spinevolution program) (31) shown are the selected
regions of the 1H NMR spectra for protons belong to C5, C2, and C3.
tion is mediated by an enzyme-assisted mechanism rather than
a spontaneous event.
HPLC-based enzyme assays were carried out in 90% D2O rather
RsU4kpxs Can Also Convert UDP-4-keto-pentose with NADH
than in H2O, and the UDP-4-keto-pentose product was puri- to UDP-xylose—When the purified Ralstonia enzyme (1 ␮g)
ϩ
fied. Fig. 6B clearly shows that the product is comprised of two was incubated with UDP-GlcA and NAD for longer period of
molecular species of UDP-4-keto-pentose: one fully protonated times, in addition to the major product of UDP-4-keto-pentose
at C5 and the other deuterated at the C5-pro-R position (Fig. (Fig. 9A, panels 5 and 6, marked by the asterisk), it yielded small
7A). Description of this mixture of enzymatic-formed species is amount of a new product with the retention time (9.3 min)
illustrated in Fig. 7B. Integration of the protons at C5 indicates corresponding to standard UDP-xylose (Fig. 9A, panel 5 and 6,
that 60% of the UDP-4-keto-pentose is deuterated (Fig. 6B). marked by arrow). The appearance of a peak at 5.47 ppm cor-
Although the pro-S H5 peak is coupled to the geminal pro-R responding to H1 of UDP-xylose was also observed when NMR
D5, the scalar coupling is less than 2 Hz and is not resolved, assays were carried out for a longer time (data not shown).
giving a singlet shifted by 11 Hz because of an isotope effect. When assays were performed with 10 ␮g of recombinant pro-
Repeating the real time NMR assay in 90% H2O produced the tein, the formation of UDP-xylose as well as UDP-4-keto-pen-
normal fully protonated product (Fig. 8) as expected.
tose was observed (Fig. 9B), suggesting that the enzyme may
The lack of any corresponding singlet in the pro-R H5 region have dual functions.
(3.51ppm) suggests that the incorporation of the deuterated
To address whether the UDP-xylose product is directly con-
proton is stereospecific. This observation may support the verted from UDP-4-keto-pentose, the recombinant RsU4kpxs
MARCH 19, 2010•VOLUME 285•NUMBER 12
JOURNAL OF BIOLOGICAL CHEMISTRY 9037

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
different affinity for the respective
UDP-sugar but more significantly
the actual rate of catalysis (Table 1).
NMR Analysis of the Enzymatic
Product, UDP-Xyl—As described in
the previous section for the UDP-4-
keto-pentose, specific deuteration
was observed at C5 in the xylose ring
when the reaction was carried out in
90% D2O. Isolating and purifying
the deuterated UDP-xylose prod-
uct and subsequent analysis via
900-MHz NMR spectrometer
(supplemental Fig. S2 and Table S1)
allowed a definitive assignment of
the pro-R H5 (equatorial) and pro-S
H5 (axial) protons. Although the
signals from the geminal H5 pro-
tons are almost fully overlapped and
typically are reported at a single
chemical shift (18, 31), at high fields
they can be resolved enough to be
identified. Fig. 11 (panel 1) shows
the region of the proton spectrum of
UDP-xylose containing the pro-R
equatorial H5 (H5e, ␦3.713, J5e,4
ϭ
6.5Hz, J5e,5a ϭ 11.3Hz), the pro-S
axial H5 (H5a, ␦3.699, J5a,4 ϭ 9.5Hz,
FIGURE 9. RsU4kpxs is a bifunctional enzyme and converts UDP-GlcA to UDP-4-keto-pentose and subse-
quently to UDP-xylose. A, purified recombinant RsU4kpxs (1 ␮g) was mixed under normal assay conditions
with UDP-GlcA, buffer, and NADϩ at 37 °C for 40 min (panel 4), or 6 h (panel 5 and 6). The reactions were also
performed without NADϩ for 6 h (panel 3). As a control, the corresponding column-purified protein isolated
from cells expressing empty vector was incubated with UDP-GlcA and NADϩ for 6 h (panel 2). The reaction
products were separated on a C18 column (Agilent prep-C18 scalar), and chromatograms are shown. UDP-4-
keto-pentose is marked by an asterisk (panel 4 and 5), and the new peak (eluted at 9.3 min, as UDP-xylose) is
marked by an arrow (panel 5 and 6). Panel 6 is an expanded view of panel 5 between 8 and 10 min. The identity
of the small peak eluted at 9.5 min (marked by **, panel 6) is UDP and was confirmed by 1H NMR (data not
shown). B, purified recombinant RsU4kpxs (10 ␮g) was mixed with UDP-GlcA, buffer, and NADϩ at 37 °C for 15
min (panel 2), 45 min (panel 3), or 90 min (panel 4). The reaction products were separated on a C18 column
(Agilent prep-C18), and chromatograms are shown. UDP-4-keto-pentose is marked by an asterisk (panels 2–4),
and the peak for UDP-xylose is marked by an arrow (panels 3 and 4).
J5a,5e ϭ 11.3Hz) and H3 (␦3.661,
J3,4 ϭ J3,2 ϭ 9.5). Fig. 11 (panel 2)
shows the spectrum of the mixture
of protonated and deuterated enzy-
matic products, with additional
signal for pro-S axial proton
(␦3.681, J5a,4 ϭ 9.5Hz, J5a,DϽ 2Hz,
marked by an asterisk) caused by
the deuterated form of the C5
pro-R position. A simulation of
the spectrum (Spinevolution pro-
(10 ␮g) was further incubated with UDP-4-keto-pentose iso-
lated from C18 column (Fig. 10). The conversion to UDP-xylose
is clearly enzymatic (Fig. 10, panel 4) and requires exogenous
NADH (Fig. 10, panel 4). NADH is required for the enzymatic
reduction step of UDP-4-keto-pentose, resulting in the forma-
gram) (32) is shown in Fig. 11 (panel 3). It is clear that the
additional doublet of H5 geminal to the deuterated D5 (Fig.
11, panel 3, H5a(d)) has a large scalar coupling (9.5 Hz) to
H4, which is consistent with its axial configuration, and is
isotopically shifted upfield by a similar amount observed in
the deuterated UDP-4-keto-pentose.
ϩ
tion and release of NAD and UDP-xylose (Fig. 1B). The analog
of NADH, NADPH, cannot substitute for NADH (data not
shown). Kinetics analyses of UDP-xylose synthase activity of
RsU4kpxs are summarized in Table 1, with Km values of 152.9
␮M (UDP-4-keto-pentose) and 94.2 ␮M (NADH); with Vmax
values (␮M minϪ1) of 3.6 (UDP-4-keto-pentose) and 3.9
(NADH) and with kcat/Km values (sϪ1 mMϪ1) of 0.081 (UDP-4-
keto-pentose) and 0.145 (NADH). The relatively lower activity
of converting UDP-4-keto-pentose to UDP-xylose cannot be
explained by the affinity to the cofactor because the Km values
DISCUSSION
We have described the cloning and biochemical character-
ization of a bi-functional UDP-4-keto-pentose/UDP-xylose
synthase from the bacterial plant pathogen R. solanacearum str.
GMI1000. The lower catalytic efficiency of the enzyme to form
UDP-xylose from UDP-4-keto-pentose is consistent with the
lower level of xylosyl residues found on Ralstonia LPS, when
compared with the 4-amino-4-deoxy-L-arabinose (L-Ara4N)
residues (33).
ϩ
for NAD and NADH are similar (113 and 94 ␮M, respectively).
However, the 150-fold difference in catalytic efficiency (kcat
/
Recent carbohydrate analyses of the R. solanacearum LPS
Km) between the two activities of the enzyme toward UDP- (composed of lipid A, a core oligosaccharide and an O-polysac-
GlcA (12.8) and UDP-4-keto-pentose (0.081) reflects the partly charide) reveal the occurrence of L-Ara4N linked to a heptose
9038 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 12•MARCH 19, 2010

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
FIGURE 11. 900-MHz proton NMR spectra of the selectively deuterated
UDP-xylose. ShownareregionsoftheprotonspectraofUDP-xyloseshowing
selective deuteration at the equatorial (pro-R) H5 position. Panel 1 shows a
spectrum (from 3.64 to 3.74ppm) of UDP-xylose that is fully protonated (C5-
He/Ha). Panel 2 shows a spectrum of UDP-xylose (C5-He/Ha and C5-De/Ha mix-
ture) isolated from the enzyme reaction in 90% D2O. Panel 3 shows a simu-
lated spectrum of the mixture of fully protonated and selectively deuterated
UDP-xylose; H5e and H5a are the equatorial and axial protons at C5, respec-
tively; H5a(d) is the axial proton where the equatorial position is deuterated;
the doublet caused by the deuterated form is marked with an asterisk. All of
the above spectra were collected via 900-MHz 1H NMR at 25 °C.
genome of R. solanacearum, it is likely that RsU4kpxs is the only
protein that provides UDP-xylose in this organism along with
UDP-4-keto-pentose.
The operon comprising RsU4kpxs in Ralstonia is similar yet
distinct from a similar operon in E. coli and Salmonella enterica
(supplemental Fig. S3B). In the latter, a gene cluster encoding
several proteins (supplemental Fig. S3) involved in producing
undecaprenyl-4-deoxy-4-formamido-L-arabinose, a key inter-
mediate in the biosynthesis of L-Ara4N-modified LPS, has been
discovered (36, 37). Some of these gene products have been
biochemically characterized (19, 38, 39). In E. coli, one single
polypeptide named ArnA is comprised of two domains: N-ter-
minal formyl-transferase domain and C-terminal decarboxyl-
ase domain. This polypeptide has the ability to interconvert
UDP-GlcA to UDP-4-keto-pentose (also named UDP-L-
FIGURE 10. The conversion of UDP-4-keto-pentose to UDP-xylose by
RsU4kpxs requires exogenous NADH. UDP-4-keto-pentose was enzymati-
cally produced (panel 1) under normal assay condition; column-purified (see
expanded view in panel 2) and used in the following assays. Purified recom-
binant RsU4kpxs (10 ␮g) was incubated with 0.4 mM UDP-4-keto-pentose in
thepresenceof0.5mM NADϩ (panel3)or0.5mM NADH(panel4)at37 °Cfor30
min. As a control, the corresponding column-purified protein isolated from
cells expressing empty vector was incubated with UDP-4-keto-pentose and
NADH for 30 min (panel 5). Reaction products were separated on C18 column
(Agilent prep-C18 scalar), and chromatograms are shown. The enzymatic
product (panel 4) was marked by an arrow, eluted at the same retention time
as authentic UDP-xylose, collected, and analyzed by 1H NMR. The identity of
the peak marked by an arrow is UDP-xylose as determined by NMR (data not
shown).
Ara4O; supplemental Fig. S3C, panel 1) and, subsequently after
ArnB function, converts the product to UDP-␤-4-deoxy-4-for-
mamido-L-arabinose (19, 39). In R. solanacearum, however, the
“equivalent E. coli ArnA gene” is split to two separate gene
products: the RsU4kpxs characterized in this report and
a
second gene likely encodes the formyl-transferase
(supplemental Fig. S3B). We thus proposed that the Ralstonia
RsU4kpxs gene was likely diverged from the ArnA and further
acquired dual enzymatic roles: The first is to produce UDP-4-
keto-pentose as precursor for the biosynthesis of L-Ara4N
modified core oligosaccharide. The second role is to form UDP-
residue of the core oligosaccharide (33), as well as a trace xylose where it can serve as precursor for synthesis of xylose
amount of a xylosyl residue linked to a rhamnose of the O-poly- containing O-polysaccharide (supplemental Fig. S3C, panel 2).
saccharide (33–35) (supplemental Fig. S3A). Therefore, the To determine the in vivo function of RsU4kpxs, we have tried to
presence of an enzyme activity of RsU4kpxs that produces disrupt the expression of the gene. However, numerous
UDP-xylose precursors in R. solanacearum is consistent with attempts to knock out RsU4kpxs gene in R. solanacearum
the presence of xylose modified LPS in that organism. Because turned out to be unsuccessful, suggesting that this gene is crit-
no UDP-GlcA decarboxylase (Uxs) homologs were found in the ical for this organism.
MARCH 19, 2010•VOLUME 285•NUMBER 12
JOURNAL OF BIOLOGICAL CHEMISTRY 9039

UDP-4-keto-pentose/UDP-xylose Synthase in R. solanacearum
10. Knirel, Y. A., Paramonov, N. A., Vinogradov, E. V., Shashkov, A. S., Dmi-
A fundamental question regarding the evolution of eukary-
otic Uxs and Uaxs enzymes is when their functions di-
verged from each other (see two possible models in
supplemental Fig. S4). Based on blast comparisons and phylo-
genetic analysis, the UXS clade includes eukaryote (animal,
fungus, and plant) Uxs and bacterial Uxs homologs (clade A),
triev, B. A., Kochetkov, N. K., Kholodkova, E. V., and Stanislavsky, E. S.
(1987) Eur. J. Biochem. 167, 549–561
11. Ma, L., Lu, H., Sprinkle, A., Parsek, M. R., and Wozniak, D. J. (2007) J.
Bacteriol. 189, 8353–8356
12. Xu, X., Ruan, D., Jin, Y., Shashkov, A. S., Senchenkova, S. N., Kilcoyne, M.,
Savage, A. V., and Zhang, L. (2004) Carbohydr. Res. 339, 1631–1636
whereas the UAXS clade has only plant members. The plant 13. Forsberg, L. S., and Carlson, R. W. (2008) J. Biol. Chem. 283, 16037–16050
14. Hwang, H. Y., and Horvitz, H. R. (2002) Proc. Natl. Acad. Sci. U.S.A. 99,
14218–14223
UAXS is more closely related to the bacterial ArnA and
Rs4Ukpxs, than it is to the plant UXS as shown in Fig. 2A. These
results imply that plant UAXS did not evolve from a plant UXS,
although they both use the same substrates and formed the
same intermediates. Several eubacteria, including cyanobacte-
ria, have UXS homologs with more sequence identity to the
plant UXS (E-value 1e-129) than to the plant UAXS, bacterial
ArnA homologs, and Clade B U4kpxs proteins. These analyses
suggest that an ancestral UXS/UAXS arose in early prokaryotes
(see model 1) and then separated into two enzymes (early bac-
terial UXS and UAXS) with distinct functions. The early bacte-
rial UXS gave rise to the current UXS homologs found in bac-
teria, animals, fungi, and plants; the early bacterial UAXS then
gave rise to the current plant Uaxs, bacterial ArnA homologs,
and clade B proteins. During evolution, the plant UAXS further
acquired a different function than RsU4kpxs, generating both
UDP-apiose and UDP-xylose via the same UDP-4-keto-pen-
tose intermediate. When the early plants acquired both the
UAXS and UXS gene from ancestral bacteria remains
uncertain.
15. Mølhøj, M., Verma, R., and Reiter, W. D. (2003) Plant J. 35, 693–703
16. Guyett, P., Glushka, J., Gu, X., and Bar-Peled, M. (2009) Carbohydr. Res.
344, 1072–1078
17. Gu, X., Wages, C. J., Davis, K. E., Guyett, P. J., and Bar-Peled, M. (2009)
J. Biochem. 146, 527–534
18. Harper, A. D., and Bar-Peled, M. (2002) Plant Physiol. 130, 2188–2198
19. Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. (2002) J. Biol. Chem. 277,
2886–2896
20. Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller,
W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402
21. Katoh, K., Kuma, K., Toh, H., and Miyata, T. (2005) Nucleic Acids Res. 33,
511–518
22. Guindon, S., and Gascuel, O. (2003) Syst. Biol. 52, 696–704
23. Gu, X., and Bar-Peled, M. (2004) Plant Physiol. 136, 4256–4264
24. Mohnen, D., Bar-Peled, M., and Somerville, C. R. (2008) in Biosynthesis of
Plant Cell Walls (Himmel, M., ed) pp. 1–54, Blackwell Publishing, Oxford
25. Rao, S. T., and Rossmann, M. G. (1973) J. Mol. Biol. 76, 241–256
26. Weirenga, R. K., Terpstra, P., and Hol, W. G. (1986) J. Mol. Biol. 187,
101–107
27. Tello, M., Rejzek, M., Wilkinson, B., Lawson, D. M., and Field, R. A. (2008)
Chembiochem. 9, 1295–1302
28. Melanc¸on, C. E., 3rd, Hong, L., White, J. A., Liu, Y. N., and Liu, H. W.
(2007) Biochemistry 46, 577–590
Acknowledgments—We thank Dr. Russell Malmberg and Malcolm
O’Neil for constructive comments on the manuscript.
29. Williams, G. J., Breazeale, S. D., Raetz, C. R., and Naismith, J. H. (2005)
J. Biol. Chem. 280, 23000–23008
30. Gatzeva-Topalova, P. Z., May, A. P., and Sousa, M. C. (2004) Biochemistry
43, 13370–13379
REFERENCES
31. Pauly, M., Porchia, A., Olsen, C. E., Nunan, K. J., and Scheller, H. V. (2000)
Anal. Biochem. 278, 69–73
1. Go¨tting, C., Kuhn, J., Zahn, R., Brinkmann, T., and Kleesiek, K. (2000) J.
Mol. Biol. 304, 517–528
32. Veshtort, M., and Griffin, R. G. (2006) J. Magn. Reson. 178, 248–282
33. Zdorovenko, E. L., Vinogradov, E., Wydra, K., Lindner, B., and Knirel, Y. A.
(2008) Biomacromolecules 9, 2215–2220
2. Kuhn, J., Go¨tting, C., Schno¨lzer, M., Kempf, T., Brinkmann, T., and
Kleesiek, K. (2001) J. Biol. Chem. 276, 4940–4947
3. Bakker, H., Oka, T., Ashikov, A., Yadav, A., Berger, M., Rana, N. A., Bai, X.,
Jigami, Y., Haltiwanger, R. S., Esko, J. D., and Gerardy-Schahn, R. (2009)
J. Biol. Chem. 284, 2576–2583
34. Kocharova, N. A., Knirel, Y. A., Shashkov, A. S., Nifant’ev, N. E., Kochet-
kov, N. K., Varbanets, L. D., Moskalenko, N. V., Brovarskaya, O. S., Muras,
V. A., and Young, J. M. (1993) Carbohydr. Res. 250, 275–287
35. Varbanets, L. D., Vasil’ev, V. N., and Brovarskaia, O. S. (2003) Mikrobiolo-
giia 72, 19–25
4. Cherniak, R., and Sundstrom, J. B. (1994) Infect. Immun. 62, 1507–1512
5. Vaishnav, V. V., Bacon, B. E., O’Neill, M., and Cherniak, R. (1998) Carbo-
hydr. Res. 306, 315–330
36. Gunn, J. S., Lim, K. B., Krueger, J., Kim, K., Guo, L., Hackett, M., and Miller,
S. I. (1998) Mol. Microbiol. 27, 1171–1182
6. Klutts, J. S., and Doering, T. L. (2008) J. Biol. Chem. 283, 14327–14334
7. O’Neill, M., and York, W. S. (2003) in The Plant Cell Wall: Annual Plant
Review (Rose, J. K. C., ed) pp. 1–54, Blackwell Publishing and CRC Press,
Oxford
37. Raetz, C. R., and Whitfield, C. (2002) Annu. Rev. Biochem. 71, 635–700
38. Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. (2003) J. Biol. Chem. 278,
24731–24739
8. Mohnen, D. (2008) Curr. Opin. Plant Biol. 11, 266–277
9. Shashkov, A. S., Arbatsky, N. P., Cedzynski, M., Kaca, W., and Knirel, Y. A.
(1999) Carbohydr. Res. 319, 199–203
39. Breazeale, S. D., Ribeiro, A. A., McClerren, A. L., and Raetz, C. R. (2005)
J. Biol. Chem. 280, 14154–14167
9040 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 12•MARCH 19, 2010

Enzymology:
Identification of a Bifunctional
UDP-4-keto-pentose/UDP-xylose Synthase
in the Plant Pathogenic Bacterium
Ralstonia solanacearum Strain GMI1000, a
Distinct Member of the 4,6-Dehydratase
and Decarboxylase Family
Xiaogang Gu, John Glushka, Yanbin Yin,
Ying Xu, Timothy Denny, James Smith,
Yingnan Jiang and Maor Bar-Peled
J. Biol. Chem. 2010, 285:9030-9040.
doi: 10.1074/jbc.M109.066803 originally published online January 29, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M109.066803
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/2010/01/29/M109.066803.DC1.html
This article cites 37 references, 17 of which can be accessed free at
http://www.jbc.org/content/285/12/9030.full.html#ref-list-1