JOURNAL OF BACTERIOLOGY, Mar. 2009, p. 1565–1573
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 5
Cloning and Characterization of Uronate Dehydrogenases from Two
Pseudomonads and Agrobacterium tumefaciens Strain C58?‡
Sang-Hwal Yoon, Tae Seok Moon, Pooya Iranpour,† Amanda M. Lanza, and Kristala Jones Prather*
Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Received 28 April 2008/Accepted 30 November 2008
Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas
putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel comple-
mentation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon
source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli
by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were
evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this
manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes
in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes.
Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized.
For all three enzymes, the turnover number (kcat) with glucuronate as a substrate was higher than that with
galacturonate; however, the Michaelis constant (Km) for galacturonate was lower than that for glucuronate.
The A. tumefaciens enzyme was found to have the highest rate constant (kcat? 1.9 ? 102s?1on glucuronate),
which was more than twofold higher than those of both of the pseudomonad enzymes.
Aldohexuronate catabolism in bacteria is reported to involve
two different pathways, one initiating with an isomerization
step and the other with an oxidation step. In the isomerization
pathway, aldohexuronate (glucuronate and galacturonate) is
isomerized to ketohexuronate by uronate isomerase and ulti-
mately degraded to pyruvate and 3-phosphoglyceraldehyde.
The isomerization pathway has been previously reported to
occur in bacteria, including Escherichia coli (7), Erwinia caro-
tovora (18), Erwinia chrysanthemi (15), Klebsiella pneumoniae
(9, 23), and Serratia marcescens (28). In the oxidation pathway,
aldohexuronate is oxidized to aldohexarate by uronate dehy-
drogenase (Udh) and further catabolized to pyruvate (2, 5, 7,
9, 18, 19, 24). Uronate dehydrogenase, the key enzyme of this
pathway, has been investigated in two plant pathogen bacteria,
Pseudomonas syringae and Agrobacterium tumefaciens. To date,
only limited studies pertaining to the properties of Udh have
been reported in the literature (3, 6, 38, 43), and no sequence
has yet been identified. Udh is classified as an NAD-linked
oxidoreductase (EC 184.108.40.206), with a total molecular weight of
about 60,000. It is a homodimer composed of two subunits with
molecular weights of about 30,000 each (38). Udh is a ther-
mally unstable, reversible enzyme, with an optimum pH of
about 8.0 (3, 6, 38).
In E. coli MG1655 that has the isomerization pathway for
aldohexuronate catabolism, glucuronate is transported by an
aldohexuronate transporter encoded by exuT and converted to
fructuronate by uronate isomerase, encoded by uxaC (22, 30)
(Fig. 1). Fructuronate is transferred to the Entner-Doudoroff
pathway to be utilized as an energy source via 2-keto-3-deoxy-
6-phospho-gluconate (7, 27, 31, 32). Therefore, E. coli
MG1655 with a uxaC deletion cannot use glucuronate as a
carbon source. In this strain, glucarate is converted to 5-keto-
4-deoxy-D-glucarate by D-glucarate dehydratase, encoded by
gudD, and then transferred to glycolysis via pyruvate or 2-phos-
phoglycerate (27, 33). Recently, a number of bacterial genome
sequences have been published, including those of the Udh-
containing P. syringae pv. tomato strain DC3000 and A. tume-
faciens strain C58 (4, 10). A shotgun library of P. syringae was
constructed to identify the gene encoding Udh. Screening for
Udh was conducted in E. coli MG1655 ?uxaC. Since uronate
dehydrogenase converts glucuronate to glucarate, uxaC dele-
tion strains of E. coli harboring the shotgun library of P. syrin-
gae that can grow in a minimal medium containing glucuronate
as a sole carbon source may carry the gene encoding Udh (Fig.
1). Once an initial Udh is identified from P. syringae, a BLAST
homology search may lead to the identification of Udhs from
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. Strains, plasmids, and
primer sequences used in this study are indicated in Table 1. Media and chemical
reagents were purchased from Sigma (St. Louis, MO) or BD Biosciences (San
Jose, CA). P. syringae pv. tomato strain DC3000 was used as the source of the
genomic library and was donated by Frederick Ausubel of Massachusetts Gen-
eral Hospital. P. syringae was grown in Luria-Bertani (LB) medium with 50 ?g/ml
rifampin (rifampicin) at 30°C. Pseudomonas putida KT2440 (ATCC 47054) was
purchased from the American Type Culture Collection (ATCC, Manassas, VA)
and grown in LB medium at 30°C. E. coli strains were grown in 2YT medium (16
g tryptone, 10 g yeast extract, and 10 g sodium chloride per liter) at 37°C. As
required, ampicillin and kanamycin were added to the medium at 100 and 25
?g/ml, respectively. Escherichia coli DH10B (F?mcrA ?(mrr-hsdRMS-mcrBC)
?80lacZ?M15 ?lacX74 recA1 endA1 araD139 ?(ara-leu)7697 galU galK ??rpsL
nupG) was used as the host strain for the genomic library as well as for subclon-
ing of screened genes (Invitrogen Corp., Carlsbad, CA). E. coli MG1655 ?uxaC
* Corresponding author. Mailing address: Department of Chem-
ical Engineering, Room 66-458, Massachusetts Institute of Tech-
nology, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone:
(617) 253-1950. Fax: (617) 258-5042. E-mail: email@example.com.
† Present address: The University of Texas Health Science Center at
San Antonio, San Antonio, TX.
‡ Supplemental material for this article may be found at http://jb
?Published ahead of print on 5 December 2008.
was provided by F. R. Blattner of the E. coli Genome Project at University of
Wisconsin—Madison. For M9 minimal agar, 22 mM glucose, glucuronate, or
glucarate was used as a carbon source. Plasmid vectors pTrc99A and pTrc99SE
were used for construction of the genomic library and as an expression vector for
candidate genes, respectively (Table 1). The plasmid pTrc99SE was donated by
Seon-Won Kim at Gyeongsang National University, Korea. pBluescript (Invitro-
gen, Carlsbad, CA) was used as a general cloning vector.
Genomic DNA preparation and construction and screening of P. syringae
genomic library. Genomic DNA preparation and general cloning procedures
were carried out as described by Sambrook and Russell (35). The genomic DNA
of A. tumefaciens strain C58 was purchased from the ATCC (ATCC number
33970D). Restriction enzymes and T4 ligase were purchased from New England
Biolabs (Beverly, MA). P. syringae genomic DNA was partially digested with
BfuCI and then loaded onto a 0.8% agarose gel. Fragments of 2 to 6 kb were
purified from the gel and then ligated to pTrc99A with dephosphorylated BamHI
ends. After ligation for 2 days at 4°C, the reaction mixtures were used to
transform E. coli DH10B. Successful transformant clones were collected and
pooled from agar plates, followed by storage at ?80°C. Plasmid pools isolated
from the colony pools were used to transform E. coli MG1655 ?uxaC to screen
for Udh activity. Transformed strains were cultured on M9 minimal agar plates
with 22 mM glucuronate for 4 days at 30°C. Surviving clones from plates were
screened by purifying and sequencing their plasmids. The sequencing results
were compared with the genome sequence of P. syringae pv. tomato strain
DC3000, as reported in GenBank (accession number NC_004578 [http://www
Construction of expression plasmid vectors containing udh genes. PCR am-
plification was carried out using Pfu Turbo AD as described by the manufacturer
(Stratagene, La Jolla, CA). The three candidate genes of iolE, iolB, and
PSPTO_1053 were each amplified from the genomic DNA using primers as listed
in Table 1. PCR products were blunt-end ligated to EcoRV-digested pBluescriptII,
resulting in pBiolE, pBiolB, pBiolEB, and pB1053, which were each sequenced
to confirm their identities. iolE, iolB, and iolEB were each cleaved by digestion
with EcoRI and SalI and then ligated to pTrc99A digested by the same enzymes
to construct pTiolE, pTiolB, and pTiolEB, respectively. PSPTO_1053 from
pB1053 was cleaved by digestion with NcoI and SacI and then ligated to pTrc99A
digested by the same enzymes, resulting in pT1053.
Putative udh genes from genomic DNA of A. tumefaciens, P. putida, and P.
syringae were amplified using the primer pairs ATudh2-F/ATudh-R, PPudh-F/
PPudh-R, and PSudh-F/1053-R, respectively (Table 1). PCR products were
blunt-end ligated to pBluescriptII digested with EcoRV, resulting in plasmids
pBATudh2, pBPPudh, and pBPSudh. To construct plasmids pTATudh2, pTPPudh,
and pTPSudh, the corresponding genes were excised with EcoRI and SacI from
pBATudh2, pBPPudh, and pBPSudh, respectively, and were inserted into the
same sites of pTrc99SE.
Protein purification and determination of kinetic parameters. The udh genes
from genomic DNA of A. tumefaciens, P. putida, and P. syringae were amplified
using primers ATuEQ-F/R, PPuEQ-F/R, and PSuEQ-F/R (Table 1), and the
PCR products were digested with SacI and HindIII and inserted into the same
sites of pET21b containing a six-His tag to construct pETATu, pETPPu, and
pETPSu, respectively (Table 1). These plasmids were used to transform E. coli
BL21(DE3) for use for protein expression. The recombinant E. coli BL21 strains
were cultivated at 30°C and 250 rpm for 6 h after IPTG (isopropyl-?-D-thioga-
lactopyranoside) induction. Protein purification was carried out using the Pro-
Bond purification system as described by the manufacturer (Invitrogen Corp.,
Carlsbad, CA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed as described by Sambrook and Russell (35). En-
zyme activities on substrates of purified proteins were measured by monitoring
initial NADH generation at 340 nm and room temperature. Kinetic analysis on
glucuronate and galacturonate was carried out using 0 to 10 mM glucuronate or
galacturonate and 1.2 mM NAD?in 100 mM Tris-HCl, pH 8.0. Kinetic analysis
on NAD?was performed using 0 to 2 mM NAD?and 10 mM glucuronate in 100
mM Tris-HCl, pH 8.0. A series of enzymatic assays were conducted to estimate
the initial activity as a function of starting substrate concentration. These data
were used to fit the parameters of the Michaelis-Menten kinetic model, kcatand
Km, by nonlinear least-squares regression. Nonlinear least-squares regression
analyses were performed via the Gauss-Newton method as implemented using
the intrinsic nlinfit function of the Matlab software program.
LC-MS and circular dichroism (CD) analysis for determination of glucarate
produced from glucuronate by Udh. The reaction mixture for the production of
glucarate from glucuronate by Udh consisted of 20 mM glucuronate, 21.6 mM
NAD?, 40 mM sodium phosphate buffer, pH 8.0, and bacterial lysate prepared
as described above. The enzyme reaction was performed by the addition of either
crude lysate or purified proteins to the reaction mixture and incubation at room
temperature for 60 min, and this was stopped by the addition of 1 M sodium
hydroxide. Glucarate was separated from the reaction mixture by using a column
packed with boronic acid affinity gel (Affi-Gel boronate gel; Bio-Rad Laborato-
ries, Hercules, CA) which is able to bind to the coplanar adjacent cis-hydroxyl
groups of glucarate (29). Glucuronate cannot bind to the gel due to its trans-diol
groups. After the Affi-Gel column was loaded with reaction mixture, the column
was washed with 80 mM potassium phosphate–20 mM boric acid buffer (pH 7.0),
and then glucarate was eluted by the addition of 0.1 M HCl. The eluent was
neutralized by the addition of 5 M NaOH then analyzed by liquid chromatog-
raphy-mass spectrometry (LC-MS) using an Agilent 1100 series LC/MSD instru-
ment (Agilent Technologies) equipped with an Aminex HPX-87H column (300
by 7.8 mm; Bio-Rad Laboratories, Hercules, CA) and an electron spray ioniza-
tion detector. Mass spectra were obtained in both the positive and negative ion
detection modes. Trifluoroacetic acid (0.1% [vol/vol]), pH 2.0, was used as the
mobile phase at a flow rate of 0.5 ml/min at room temperature.
FIG. 1. Catabolism of glucuronate and glucarate in bacteria. Glucuronate consumption is prevented by knockout of the uxaC gene. The
presence of uronate dehydrogenase in a uxaC knockout enables growth of E. coli on glucuronate.
1566 YOON ET AL.J. BACTERIOL.
The stereochemistry of glucarate formed from glucuronate was confirmed by
comparing its CD spectrum with that of an authentic glucarate standard. CD was
performed on a model 202 CD spectrometer (Aviv Biomedical, Lakewood, NJ).
Reaction mixtures contained 20 mM glucuronic acid, 7 mM NAD?, 100 mM
potassium phosphate buffer (pH 8.0), and the purified enzymes prepared as
described above. Glucarate was separated from glucuronate by using boronic
acid affinity gel as described above.
Computational analysis including sequence identification and alignment
and metabolites. DNA sequences for P. syringae, P. putida, and A. tumefaciens were
obtained from the National Center for Biotechnology Information (NCBI; http:
//www.ncbi.nlm.nih.gov/), with accession numbers NC_004578, NC_002947, and
NC_003063, respectively. Homology and conserved domain searches were per-
formed using the NCBI BLAST algorithm. Sequence management and alignment
were carried out using Vector NTI software (Invitrogen, Carlsbad, CA). Alignment
and phylogenetic analyses were performed using the AlignX module of Vector NTI.
Nucleotide sequence accession numbers. The udh gene sequence from P.
syringae has been deposited in GenBank (accession number EU377538). The
corresponding genes from A. tumefaciens and P. putida were deposited with
accession numbers BK006462 and BK006380, respectively.
Cloning of the udh gene from Pseudomonas syringae. The
screen established to identify the gene corresponding to
Udh activity in P. syringae utilized a mutant strain of E. coli
MG1655. The deletion of uxaC prevents growth on glucuro-
nate while permitting the strain to retain the ability to grow on
glucarate as a sole carbon source. Since Udh catalyzes the
conversion of glucuronate to glucarate (3, 38), E. coli MG1655
?uxaC clones harboring udh genes from a P. syringae genomic
library should grow on glucuronate as the sole carbon source.
E. coli DH10B and pTrc99A were used as the host strain and
TABLE 1. Strains, plasmids, and primers used in this study
Strain, plasmid, or primer Descriptionb
Reference or source
Pseudomonas syringae pv.
tomato strain DC3000
Pseudomonas putida KT2440
Escherichia coli DH10B
Wild typeFrederick Ausubel
F?mcrA ?(mrr-hsdRMS-mcrBC) ?80lacZ?M15 ?lacX74 recA1 endA1
araD139 ?(ara-leu)7697 galU galK ??rpsL nupG
Wild type with deletion of the uxaC gene, encodes D-glucuronate isomerase
Escherichia coli MG1655 ?uxaC
Escherichia coli BL21(DE3)
?) gal dcm (DE3)
lac promoter, ColE1 origin, ampicillin resistant, lacZ
trc promoter, pBR322 origin, ampicillin resistant, lacIq
T7 promoter, ColE1 origin, ampicillin resistant, lacI
Stratagene, La Jolla, CA
pTrc99A containing RBS sequence of AGGAGGTAATAAAT
pTrc99A with iolE of P. syringae
pTrc99A with iolB of P. syringae
pTrc99A with iolE and iolB of P. syringae
pTrc99A with PSPTO_1053 of P. syringae
pTrc99SE with udh of A. tumefaciens
pTrc99SE with udh of P. putida
pTrc99SE with udh of P. syringae
pET21b with udh of A. tumefaciens
pET21b with udh of P. putida
pET21b with udh of P. syringae
aPrimer binding sites, restriction sites, and start or stop codons are indicated as letters with boldface, double underline, and single underline, respectively.
bRBS, ribosome binding site.
VOL. 191, 2009 CLONING OF URONATE DEHYDROGENASES1567
plasmid vector, respectively, for the initial construction of the
P. syringae genomic library. A plasmid library pool was pre-
pared from the E. coli DH10B clone pool and then used to
transform the ?uxaC strain. Transformed ?uxaC clones were
incubated on M9 minimal agar containing glucuronate for 4
days at 30°C.
From 10 agar plates, 28 clones were selected for further
screening, each of which contained an inserted fragment of 2 to
5 kb. From these, eight clones with different-sized inserts were
sequenced for comparison with the P. syringae genome se-
quence (GenBank accession number NC_004578). Six of these
clones included iolE, iolB, or both of them, while one clone
contained the unassigned PSPTO_1053 open reading frame.
The final clone included a chimera of the iolEB and PSPTO_1053
PCR amplified and inserted into expression vector pTrc99A,
yielding plasmids pTiolE, pTiolB, pTiolEB, and pT1053. Clones
containing these vectors were used to determine which gene cor-
responded to uronate dehydrogenase activity. E. coli MG1655,
the ?uxaC derivative, and four ?uxaC clones transformed with
the candidate genes were incubated on M9 minimal agar
containing glucuronate as the sole carbon source. Wild-type
?uxaC, and MG1655(pT1053) ?uxaC strains grew on M9-
glucuronate agar, while the MG1655(pTrc99A) ?uxaC and
MG1655(pTiolE) ?uxaC strains did not. Therefore, iolB and
PSPTO_1053 were responsible for growth on glucuronate as the
sole carbon source, identifying them as candidate udh genes.
To further discriminate between the two candidate genes,
plasmids pTiolB and pT1053 were used to transform E. coli
DH10B to express the recombinant genes. The resulting clones
were grown in LB medium with 0.1 mM IPTG. Analysis of Udh
activity in crude lysates from these two clones suggested that
the strain harboring pT1053, but not that harboring pTiolB,
exhibits Udh activity. The assay employed glucuronate as a
substrate and monitored production of NADH at 340 nm.
Consequently, it was deduced that the 828-bp PSPTO_1053
gene encoded uronate dehydrogenase. The gene is hereafter
referred to as udh and was registered in GenBank (http://www
.ncbi.nlm.nih.gov/Genbank/index.html) under accession num-
Cloning and identification of udh genes from P. putida and
A. tumefaciens. The translated protein sequence from udh from
P. syringae was analyzed using BLASTP from NCBI (http:
//www.ncbi.nlm.nih.gov/blast/) to identify putative homologues.
The Udh activity of A. tumefaciens has been studied previously
(5, 6, 43). The translation of open reading frame Atu3143 of A.
tumefaciens had the highest sequence identity from this organ-
ism (47.8%) and was considered a candidate for a homologous
Udh. Another candidate open reading frame, PP1171 of
Pseudomonas putida KT2440, was also found to have high
similarity to P. syringae Udh, with a sequence identity of 75.6%.
Atu3143 and PP1171 were PCR amplified from their respec-
tive genomes and, along with udh from P. syringae, were inte-
grated into plasmid vector pTrc99SE to create plasmids
pTATudh2, pTPPudh, and pTPSudh, respectively, for compar-
ison of relative activities of the expressed recombinant pro-
teins. Transformed DH10B clones were cultivated in LB me-
dium with or without 0.1 mM IPTG before the preparation of
crude lysates to carry out enzymatic analysis. These assays
confirmed a NAD?-consuming activity in the presence of gluc-
uronate as a substrate for the recombinant proteins of A.
tumefaciens and P. putida, similar to that previously obtained
with P. syringae. The two udh genes from A. tumefaciens and P.
putida were also deposited in GenBank under accession num-
bers BK006462 and BK006380, respectively.
Purification and characterization of recombinant Udh and
analysis of the reaction product. Enzyme reactions using crude
E. coli lysates containing the P. syringae udh gene confirmed
the presence of an activity that utilized glucuronate as a sub-
strate, with the reaction rate proportional to glucuronate con-
centration for low substrate loads (data not shown). The ac-
tivity also utilized NAD?but not NADP?as a cofactor (data
not shown). These results indicated that the substrate was
oxidized. An examination of the structure of glucuronate sug-
gests two possible points of oxidation: the conversion of an
alcohol to a ketone or the conversion of the aldehyde to car-
boxylic acid, the latter reaction producing glucarate. The dif-
ference in these two products should be evident from mass
spectra, as the former would result in a mass difference of ?2
relative to the substrate, while the latter would produce a mass
difference of ?16. To confirm the product of the enzyme re-
action as glucarate, a sample was analyzed by LC-MS. The
spectra of the eluent separated from the enzyme reaction and
a glucarate standard were in agreement, suggesting glucarate
as the product of the Udh reaction (see Fig. S1 in the supple-
Each of the three udh genes was expressed in E. coli with
six-His tags and purified to determine the kinetic parameters
of the corresponding enzymes. Purified enzymes were analyzed
by SDS-PAGE to confirm the molecular weight of the mono-
mer and estimate purity (Fig. 2). The Udh proteins of P.
syringae and P. putida both had molecular weights of approxi-
mately 30,000, which are consistent with both the translation of
the cloned gene and previous reports (38). The A. tumefaciens
Udh is slightly larger, with a molecular weight of 32,000.
FIG. 2. SDS-PAGE analysis of purified Udhs. The purified Udhs
were subjected to electrophoresis in a 12% SDS-polyacrylamide gel
under denaturing conditions. Lane 1, molecular weight markers;
lanes 2 and 3, crude extract and purified A. tumefaciens Udh of E.
coli BL21(DE3) expressing pETATu; lanes 4 and 5, crude extract
and purified P. putida Udh of E. coli BL21(DE3) expressing
pETPPu; lanes 6 and 7, crude extract and purified P. syringae Udh of
E. coli BL21(DE3) expressing pETPSu. Molecular masses (in kDa,
equivalent to molecular weights in thousands) are shown to the left.
The purified Udhs are indicated by the arrows.
1568YOON ET AL.J. BACTERIOL.
The purified preparations were used to determine the ki-
netic parameters, kcatand Km, for each of the enzymes. Both
glucuronate and galacturonate were used as substrates, and the
NAD?cofactor concentration was also varied to determine the
corresponding Km(Table 2). Measurements of kcatobtained by
varying the cofactor concentration were within 20% of the
values obtained using glucuronate as the substrate (data not
shown). In all cases, the kcatfor glucuronate was higher than
that for galacturonate. The highest rate constant was found for
the A. tumefaciens enzyme utilizing glucuronate as the sub-
strate (kcat? 1.9 ? 102s?1), which was more than twofold
higher than the rate for the Pseudomonas enzymes. However,
the Michaelis (affinity) constant was lower for galacturonate in
all cases, with the lowest Km, 0.04 mM, found for the P. syringae
enzyme utilizing galacturonate as the substrate. The first-order
rate constants (kcat/Km) were highest for galacturonate as sub-
strate, with the largest difference between glucuronate and
galacturonate observed for P. syringae.
The responses of the enzyme activities to changes in pH and
temperature were also investigated (Fig. 3). A pH optimum of
8.0 was observed for both the A. tumefaciens and P. syringae
enzymes, although the activities were relatively unchanged be-
tween pH ?7 and pH ?8 for P. syringae Udh (Fig. 3a). This pH
behavior is consistent with previous reports for P. syringae Udh
(3). The P. putida enzyme exhibited highest activity at pH ?7.0.
In general, enzyme activities varied approximately 10% be-
tween pH ?5 and pH ?8, with significant drops in activity
observed for pH values greater than 8 for all three enzymes.
The impact of temperature was evaluated in two ways. First,
the thermal stability was examined by exposing enzyme prep-
arations to various temperatures for 30 min and then perform-
ing the enzyme assay under standard conditions. The A. tume-
faciens Udh was found to exhibit a significantly higher thermal
stability than either of the Pseudomonas enzymes (Fig. 3b).
The activity remained near 80% of the maximum after expo-
sure of the A. tumefaciens preparation to 37°C, while the cor-
responding activities for both of the other enzymes were below
20% of the maximum. The stability profiles for the two Pseudo-
monas enzymes were similar to one another. Finally, enzyme
activity was evaluated for assays conducted under increasing
temperatures. These activities followed a general trend of in-
creasing with increasing temperatures between 4 and 42°C,
which is consistent with an Arrhenius-type dependence of the
catalytic rate constant on temperature (Fig. 3c).
For the final characterization of the products of these reac-
tions, the boronic acid affinity gel was used to isolate the
putative glucarate produced from all three enzymes in in vitro
reactions using purified proteins. Samples of the three prod-
ucts were then subjected to CD analysis to examine the stereo-
chemistry of the compounds. All three spectra were in agree-
ment with a glucarate standard, confirming the identity of the
product as glucaric acid and the identity of the three genes as
those encoding uronate dehydrogenases (data not shown).
Udh catalyzes the first step of an oxidation pathway for
aldohexuronate catabolism in bacteria. For bacteria, only lim-
ited studies of Udh in P. syringae and A. tumefaciens have been
reported. Moreover, Udh has been even more rarely studied in
eukaryotes. A Udh sequence in the wine grape Vitis vinifera has
been identified as galacturonate reductase (EC 220.127.116.11;
BRENDA accession number A1Y2Z0, GenBank accession
number DQ843600). We synthesized this gene with codon us-
age optimized for expression in E. coli (DNA 2.0, Menlo Park,
CA) and expressed the recombinant protein. However, no
TABLE 2. Turnover numbers and Michaelis constants of uronate
dehydrogenases from A. tumefaciens, P. putida, and P. syringae
1.9 ? 0.1
0.92 ? 0.14
0.37 ? 0.12
0.16 ? 0.12
0.18 ? 0.03
0.55 ? 0.03
0.30 ? 0.03
0.25 ? 0.07
0.10 ? 0.06
0.21 ? 0.02
0.74 ? 0.03
0.24 ? 0.01
0.28 ? 0.07
0.04 ? 0.01
0.17 ? 0.07
FIG. 3. Effects of pH and temperature on activities of Udhs from A. tumefaciens, P. putida, and P. syringae udh. (a) Relative activities as a
function of pH. (b) Relative activities after incubation for 30 min at indicated temperatures. (c) Relative activities as a function of assay
temperature. Squares, A. tumefaciens Udh; circles, P. putida Udh; triangles, P. syringae Udh.
VOL. 191, 2009CLONING OF URONATE DEHYDROGENASES 1569
activity related to Udh was observed when using either NAD?
or NADP?as a cofactor (data not shown). An alignment of
this sequence with the P. syringae Udh identified in the current
work reveals only 10% identity between them. We cannot rule
out the possibility that the V. vinifera enzyme could not be
functionally expressed in E. coli; however, based on the align-
ment, we conclude that the reported sequence from V. vinifera
either is not uronate dehydrogenase or is a highly divergent
version of the enzyme.
A shotgun library of P. syringae was introduced into uxaC
deletion strains of E. coli to screen for the udh gene encoding
uronate dehydrogenase, and PSPTO_1053 and iolB were iden-
tified and screened as possible Udh gene candidates. By enzy-
matic analysis, PSPTO_1053 was ultimately identified as the
udh gene encoding uronate dehydrogenase. In a uxaC deletion
mutant of E. coli, in which glucuronate catabolism is abolished,
glucuronate was converted to glucarate by uronate dehydroge-
nase and then degraded to pyruvate or 2-phosphoglycerate,
from which it can be used as an energy source (27, 33). In uxaC
deletion strains of E. coli, introduction of the iolB gene allowed
for growth on M9 agar containing glucuronate as a sole carbon
source as well, but this gene did not possess Udh activity. IolB
has previously been reported as a protein related to myo-
inositol catabolism in Bacillus subtilis and Lactobacillus casei
(41, 42). IolB belongs to the iol operon used for myo-inositol
degradation in Bacillus subtilis and converts 5-deoxy-glucuro-
nate to 2-deoxy-5-keto-D-gluconate (42). IolB of P. syringae has
about 48% homology with that of B. subtilis. The precise mech-
anism of glucuronate consumption in cells harboring IolB in
our screen is unclear. Presumably, this protein is able to con-
vert glucuronate to an analogous compound that is compatible
with E. coli metabolism.
The udh gene loci in the genomes of P. syringae, P. putida,
and A. tumefaciens are illustrated in Fig. 4. The udh loci of P.
syringae and P. putida are at about 1,150 and 1,346 kb, respec-
tively, while the udh locus in A. tumefaciens is at about 150 kb.
In A. tumefaciens, the genes Atu3140, Atu3141, Atu3142, and
Atu3145 adjacent to udh are kdgD, kduD, kduI, and kdgF,
respectively, and are related to pectin degradation. Pectin is a
heteropolysaccharide, consisting of ?-1,4-linked D-galacturo-
nate residues, which is derived from plant cell walls. Pectin
degradation and uptake by bacteria have been well researched
in studies of phytopathogenic pectobacteria, including Erwinia
chrysanthemi and Erwinia carotovora by Hugouvieux-Cotte-
Pattat et al. (12–14). In E. chrysanthemi, pectin is degraded by
genes of the kdu or kdg operon to use as an energy source. In
P. syringae and P. putida, the genes adjacent to udh are iden-
tified as TRAP (tripartite ATP-independent periplasmic) di-
FIG. 4. Loci of udh genes on chromosomes of P. syringae pv. tomato strain (str.) DC3000 (a), P. putida KT2440 (b), and A. tumefaciens strain
C58 (c). (d) Identities of adjacent genes. These loci and identities refer to the genome sequences of NC_004578 (P. syringae pv. tomato strain
DC3000), NC_002947 (P. putida KT2440), and NC_003063 (A. tumefaciens strain C58).
1570 YOON ET AL.J. BACTERIOL.
carboxylate transporters and porin. Among these genes, the
porin protein gene (PSPTO_1054 and PP_1173) is known to be
related to uptake of oligogalacturonate derived from pectin
degradation (34). Uronate dehydrogenase in plant pathogen
bacteria might therefore function in the utilization of a hexuro-
nate, derived from host plant cell wall pectin, which is subse-
quently converted to hexarate.
Alignment of the three uronate dehydrogenases from P.
syringae, P. putida, and A. tumefaciens and phylogenetic anal-
ysis of their homologs were performed (Fig. 5). The sequences
of the enzymes show two primary sequence motifs, YxxxK and
GxxGxxG, related to conserved domains (Fig. 5a). The YxxxK
motif is located between Tyr145and Lys149of P. syringae Udh
and is the primary motif of the 3-alpha/beta hydroxysteroid
dehydrogenase domain (11, 37). The GxxGxxG motif located
in the Gly18-to-Gly24region of P. syringae Udh is similar to
Rossman folds, GxxxG or Gx1-2GxxG, which have been discov-
ered in NAD?binding domains (20). In the phylogenetic anal-
ysis, the uronate dehydrogenase showed homologies with
NAD-dependent epimerase/dehydratase, nucleotide sugar epi-
FIG. 5. (a) Alignment of uronate dehydrogenase from P. syringae pv. tomato strain (str.) DC3000, P. putida KT2440, and A. tumefaciens strain
C58. For alignment, identical, conservative, and similar amino acid residues are represented as black, dark gray, and light gray blocks, respectively.
Primary sequence motifs are indicated as GxxGxxG and YxxxK. (b) Phylogenetic analysis of the uronate dehydrogenase homologues from diverse
prokaryotic and eukaryotic species. Phylogenetic analysis was performed using homologues of PSPTO_1053 of P. syringae pv. tomato strain
DC3000. Uronate dehydrogenases are indicated in bold.
VOL. 191, 2009CLONING OF URONATE DEHYDROGENASES1571
merase, 3-beta hydroxysteroid dehydrogenase/isomerase, and
short-chain dehydrogenase/reductase in archaea and bacteria,
including proteobacteria, cyanobacteria, green nonsulfur
bacteria, and gram-positive bacteria, as well as homology
with nucleotide sugar epimerase in a few eukaryotes, includ-
ing fungi, plants, and humans (Fig. 5b). The three uronate
dehydrogenases screened in this study are present in alpha-
proteobacteria and gammaproteobacteria, and their homol-
ogies are relatively close to those in the Archaea Halorubrum
lacusprofundi and Natronomonas pharaonis and the fungus
We have screened and sequenced three uronate dehydroge-
nases from A. tumefaciens, P. putida, and P. syringae that can
effectively convert glucuronate to glucarate. While this enzyme
is important for the catabolism of uronic acids in several types
of bacteria, it may also be useful in the development of bio-
synthetic pathways for the production of aldaric acids, such as
glucaric acid. Glucarate is the end product of nucleotide sugar
metabolism and is found naturally in mammals and plants (21,
39). Glucarate and its derivatives, such as glucaro-1,4-lactone,
have been studied previously as detoxifying and natural anti-
carcinogenic compounds (8, 21, 36, 39), as well as building
blocks for polymer synthesis (16). Glucarate has also been
designated as a potential “top value-added” chemical to be
produced from biomass (40). Presently, glucarate is synthe-
sized from glucose by chemical oxidation using a strong oxi-
dant such as nitric acid or nitric oxide (25). We used the udh of
P. syringae identified in this study to successfully produce glu-
caric acid from a synthetic pathway in E. coli (26).
This work was supported by the Office of Naval Research Young
Investigator Program (grant no. N000140510656). S.-H.Y. was sup-
ported by the Korea Research Foundation Grant funded by the Ko-
rean Government (MOEHRD) (KRF-2007-357-D00090). A.M.L. was
supported by a Merck Undergraduate Research Grant (Bioprocess
R&D, West Point, PA).
We are appreciative of Frederick Ausubel of the Massachusetts
General Hospital for the donation of P. syringae pv. tomato DC3000
and of Seon-Won Kim at Gyeongsang National University, Korea, for
the donation of the pTrc99SE plasmid vector. We thank Koli Taghiza-
deh, codirector of the Bioanalytical Core, Center for Environmental
Health Sciences at Massachusetts Institute of Technology, for support-
ing analysis by LC-MS.
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