Crystal structure of uronate dehydrogenase from Agrobacterium tumefaciens

Article abstract of DOI:10.1074/jbc.M111.254854

Uronate dehydrogenase from Agrobacterium tumefaciens (AtUdh) belongs to the short-chain dehydrogenase/reductase superfamily and catalyzes the oxidation of D-galacturonic acid and D-glucuronic acid with NAD⁺ as a cofactor. We have determined the crystal structures of an apo-form of AtUdh, a ternary form in complex with NADH and product (substrate-soaked structure), and an inactive Y136A mutant in complex with NAD⁺. The crystal structures suggest AtUdh to be a homohexamer, which has also been observed to be the major form in solution. The monomer contains a Rossmann fold, essential for nucleotide binding and a common feature of the short-chain dehydrogenase/reductase family enzymes. The ternary complex structure reveals a product, D-galactaro-1,5-lactone, which is bound above the nicotinamide ring. This product rearranges in solution to D-galactaro-1,4-lactone as verified by mass spectrometry analysis, which agrees with our previous NMR study. The crystal structure of the mutant with the catalytic residue Tyr-136 substituted with alanine shows changes in the position of Ile-74 and Ser-75. This probably altered the binding of the nicotinamide end of NAD⁺, which was not visible in the electron density map. The structures presented provide novel insights into cofactor and substrate binding and the reaction mechanism of AtUdh. This information can be applied to the design of efficient microbial conversion of D-galacturonic acid-based waste materials.

Full text of DOI:10.1074/jbc.M111.254854

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 31, pp. 27294–27300, August 5, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Crystal Structure of Uronate Dehydrogenase from
□
S
Agrobacterium tumefaciens*
Received for publication, April 26, 2011, and in revised form, June 2, 2011 Published, JBC Papers in Press, June 15, 2011, DOI 10.1074/jbc.M111.254854
Tarja Parkkinen‡, Harry Boer§, Janne Ja¨nis‡, Martina Andberg§, Merja Penttila¨§, Anu Koivula§, and Juha Rouvinen‡1
From the ‡Department of Chemistry, University of Eastern Finland, FI-80101 Joensuu, Finland and the §VTT Technical Research
Centre of Finland, FI-02044 VTT, Finland
Uronate dehydrogenase from Agrobacterium tumefaciens
(AtUdh) belongs to the short-chain dehydrogenase/reductase
superfamily and catalyzes the oxidation of D-galacturonic acid
way, have been found in bacteria. The isomerase pathway (in
Escherichia coli) converts D-galacturonic acid into pyruvate and
D-glyceraldehyde 3-phosphate. The oxidative pathway has been
described for Agrobacterium tumefaciens and Pseudomonas
syringae (2, 3). In this pathway, D-galacturonic acid is first oxi-
dized into meso-galactaric acid and then converted in the fol-
lowing step to ␣-ketoglutarate.
؉
and D-glucuronic acid with NAD as a cofactor. We have deter-
mined the crystal structures of an apo-form of AtUdh, a ternary
form in complex with NADH and product (substrate-soaked
structure), and an inactive Y136A mutant in complex with
؉
NAD . The crystal structures suggest AtUdh to be a homohex-
Uronate dehydrogenase (EC 1.1.1.203) is the key enzyme in
the oxidative pathway of D-galacturonic acid catabolism in bac-
teria. The enzyme catalyzes the oxidation of D-galacturonic acid
into D-galactaric acid. Uronate dehydrogenases from A. tume-
faciens (4) and P. syringae (5) have been purified and character-
ized, and the corresponding genes have also been identified (6,
7). A. tumefaciens (Rhizobium radiobacter) uronate dehydro-
amer, which has also been observed to be the major form in
solution. The monomer contains a Rossmann fold, essential for
nucleotide binding and a common feature of the short-chain
dehydrogenase/reductase family enzymes. The ternary complex
structure reveals a product, D-galactaro-1,5-lactone, which is
bound above the nicotinamide ring. This product rearranges in
solution to D-galactaro-1,4-lactone as verified by mass spec-
trometry analysis, which agrees with our previous NMR study.
The crystal structure of the mutant with the catalytic residue
Tyr-136 substituted with alanine shows changes in the position
of Ile-74 and Ser-75. This probably altered the binding of the
ϩ
genase (AtUdh)2 is specific for NAD as a cofactor but accepts
both D-galacturonic acid and D-glucuronic acid as substrates
with similar affinities. AtUdh belongs to the short-chain dehy-
drogenase/reductase (SDR) superfamily. SDR proteins are
NAD(P)(H)-dependent enzymes with a wide spectrum of sub-
strate specificities and also different enzyme classes (21). The
sequence identities between the members of the SDR family are
low, but they share a similar three-dimensional ␣/␤-structure.
To date, no structural information on uronate dehydrogen-
ase is available. Here, we present the first three-dimensional
structure of uronate dehydrogenase, namely AtUdh. We deter-
mined the crystal structures of AtUdh in the apo-form and the
ternary complex with NADH and product at 1.9 and 2.1 Å res-
olutions, respectively. In addition, we performed a site-directed
mutagenesis study of the catalytic residue Tyr-136 and deter-
؉
nicotinamide end of NAD , which was not visible in the electron
density map. The structures presented provide novel insights
into cofactor and substrate binding and the reaction mechanism
of AtUdh. This information can be applied to the design of effi-
cient microbial conversion of D-galacturonic acid-based waste
materials.
D-Galacturonic acid is the main component of pectin, a nat-
ural polymer that exists in primary cell walls of terrestrial
plants. Citrus peel and sugar beet pulp are cheap raw materials,
and both contain a large amount of pectin, which is currently
exploited mainly as cattle feed. Pectin has the potential to be an
important raw material for biotechnical conversions to fuels
and chemicals. The microbial pathways of D-galacturonic acid
catabolism have recently been described (1). Two different cat-
abolic pathways, the isomerase pathway and the oxidative path-
ϩ
mined the NAD -bound crystal structure of the inactive
mutant Y136A. This crystallographic information has enabled
us to identify the active site of the enzyme and the molecular
basis for cofactor and substrate recognition. We also propose a
structure-based mechanism for the oxidation of D-galacturonic
acid. This information can be used to improve the properties of
the enzyme, especially the substrate specificity and enzymatic
activity.
EXPERIMENTAL PROCEDURES
* This work was supported by the Academy of Finland through the Finnish
Centre of Excellence in White Biotechnology-Green Chemistry (Decision
118573).
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. S1.
The atomic coordinates and structure factors (codes 3RFT, 3RFV, and 3RFX)
have been deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
Cloning, Expression, and Purification of AtUdh—Cloning,
expression, and purification were performed as described
previously (6). Briefly, the N-terminal His6-tagged gene was
expressed in Saccharomyces cerevisiae under the control of the
□S
1 To whom correspondence should be addressed: Dept. of Chemistry, Univer- 2 The abbreviations used are: AtUdh, A. tumefaciens uronate dehydroge-
sity of Eastern Finland, P. O. Box 111, FIN-80101 Joensuu, Finland. Tel.: 358-
13-251-3318; Fax: 358-13-251-3390; E-mail: juha.rouvinen@uef.fi.
nase; SDR, short-chain dehydrogenase/reductase; DesIV, dTDP-glucose
4,5-dehydratase.
27294 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of Uronate Dehydrogenase
constitutive triose-phosphate isomerase promoter, and AtUdh processed using Bruker Daltonics Compass 1.1 for Esquire/
was purified from the yeast cell extract in a single step using HCT software. The spectra were further analyzed with Bruker
nickel-nitrilotriacetic acid chromatography.
DataAnalysis 4.0 software.
Site-directed Mutagenesis—The His6-tagged uronate dehy-
Crystallization—All crystallization experiments were per-
drogenase gene in the p2159 vector (6) was used as the template formed at room temperature using the hanging drop vapor dif-
for all mutagenesis reactions. A site-directed mutation of tyro- fusion method. The protein concentration was 4 mg/ml in 50
sine at position 136 to alanine was introduced using the mM sodium phosphate (pH 8), 150 mM NaCl, and 1 mM ␤-mer-
QuikChange site-directed mutagenesis kit from Stratagene (La captoethanol. 1 ␮l of protein solution was mixed with 1 ␮l of
Jolla, CA). The mutant was transformed into the E. coli XL1- reservoir solution and equilibrated against 0.5 ml of reservoir
Blue strain, isolated, and sequenced using an ABI PRISM 3100 solution. Crystal screening was carried out using Crystal
genetic analyzer (Applied Biosystems, Foster City, CA). Expres- ScreenTM (Hampton Research). The first crystallization condi-
sion and purification of the His6-tagged Y136A mutant were tion consisted of 2.0 M ammonium sulfate and 0.1 M sodium
carried out in S. cerevisiae using essentially the same protocol as acetate (pH 4.6). To optimize the size of the crystals, the con-
for the wild-type enzyme. The activity of the purified mutant centration of ammonium sulfate was decreased to 1.8 M. Under
enzyme was assayed on D-galacturonic acid at pH 7.5 and 2 °C this condition, crystals 0.4 ϫ 0.4 ϫ 0.4 mm in size grew within
by following the formation of NADH at 340 nm as described (6). a few days. These crystals were used for crystal structure deter-
Inhibition of AtUdh Activity—The effect of sulfate on AtUdh mination and for the apo-structure. For the soaking experi-
activity was determined by following the change in absorbance ments, a second crystallization condition was optimized
ϩ
at 340 nm in the presence of 1 ␮M to 100 mM ammonium sulfate because only half of the NAD molecule was observed in the
at 22 °C. In the assay, 1 ␮g of AtUdh was used with 4 mM D-ga- cofactor-binding site in the first crystals. The second crystalli-
ϩ
lacturonic acid and 0.5 mM NAD in 50 mM Tris-HCl (pH 7.5) zation condition consisted of 1.8 M ammonium phosphate and
containing 10 mM MgCl2.
Tris-HCl (pH 8.5), and the crystals were isomorphous with the
Size-exclusion Chromatography—Size-exclusion chroma- crystals obtained earlier. These latter conditions were also used
tography to analyze the oligomeric state of the AtUdh was per- for crystallization of the Y136A mutant.
formed at 22 °C on a Superdex 200 HR 10/30 column equili-
The heavy atom derivatives were prepared by soaking crys-
brated in 50 mM sodium phosphate and 150 mM NaCl (pH 8.0) tals in cryoprotectant solution (reservoir solution containing
and run at a flow rate of 0.5 ml/min. The column was calibrated 30% glycerol) containing 10 mM heavy atom for 10 min. For the
ϩ
with gel filtration standard proteins (GE Healthcare) giving the soaking of crystals, 5 mM NAD and 10 mM D-galacturonic acid
following elution volumes: ferritin (440,000 Da), 14.3 ml; aldol- was added to the reservoir solution. Crystals were soaked for
ase (158,000 Da), 15.3 ml; ovalbumin (44,000 Da), 16.5 ml; and ϳ10 min and transferred to cryoprotectant solution containing
ribonuclease A (13,700 Da), 18.0 ml. The void volume of the soaking solution with an additional 30% of glycerol. After a
column was determined by thyroglobulin, which eluted at 8.5 quick soak in cryoprotectant solution, the crystals were cooled
ml. A 250-␮l sample containing 29 ␮g of AtUdh was loaded in liquid nitrogen.
onto the column, and the eluate was monitored at 280 nm.
Fractions of 0.5 ml were collected, and aliquots from the frac- All crystals were frozen in liquid nitrogen after being soaked in
tions were analyzed by SDS-PAGE. cryoprotectant solution, and the data were collected at 100 K.
Data Collection and Processing and Structure Determination—
Mass Spectrometry—Electrospray ionization MS was per- European Synchrotron Radiation Facility (ESRF) beamlines
formed on a quadrupole ion trap instrument (Esquire 3000 ID14-4 and ID23-1 and DESY beamline X12 were used as x-ray
Plus, Bruker Daltonics, Bremen, Germany) operated in a nega- sources. All data sets were indexed, integrated, and scaled using
tive-ion mode. This instrument has been described in detail the program package XDS/XSCALE (9). The apo-crystals dif-
previously (8). D-Galacturonic acid was dissolved in water to fracted to a resolution of 1.9 Å. The crystals belong to the hex-
prepare a 100 mM stock solution. For electrospray ionization agonal space group P6222 (a and b ϭ 164.6 Å and c ϭ 174.8 Å),
MS, the stock solution was further diluted to 10 ␮M with ace- with a solvent content of 68% and three molecules in the asym-
tonitrile and directly electrosprayed at a flow rate of 2 ␮l/min. metric unit. The crystal structure of AtUdh was solved by the
For hydrogen/deuterium exchange, the stock solution was first SIRAS (single isomorphous replacement with anomalous scat-
diluted to 100 ␮M with D2O, allowed to stand for 15 min, and tering) method using the HKL2MAP graphical user interface
further diluted to 10 ␮M with acetonitrile-d3. For collision-in- (10). Native and derivative data sets were scaled together with
duced dissociation MS/MS spectra, the ions of interest were SHELXC, and the heavy atom positions were determined by
mass-selected and fragmented using the most appropriate frag- using SHELXD (11). SHELXE was then used to compute the
mentation amplitudes. For reaction end product analysis, a protein phases using a KAu(CN)2 derivative crystal. The result-
ϩ
mixture of 100 ␮M D-galacturonic acid and 180 ␮M NAD was ant electron density map was used to automatically fit the
prepared in 1 ml of 10 mM ammonium bicarbonate (pH 8.3), sequence using Buccaneer (12), which built a total of 796 resi-
and 2 ␮l of AtUdh (4 mg/ml) was added to the reaction mixture dues for the three molecules. The remainder of the model was
and allowed to stand at room temperature for 30 min. The manually built with COOT (13), and the refinement was subse-
reaction was stopped by diluting the reaction mixture with ace- quently performed with PHENIX (14). The structures of AtUdh
ϩ
tonitrile (1:10) and directly analyzed. For hydrogen/deuterium soaked with NAD and D-galacturonic acid at pH 8.5 and the
ϩ
exchange, 10 mM ammonium bicarbonate prepared in D2O was Y136A mutant soaked with NAD were solved by molecular
used instead. The instrument was controlled and the data were replacement using the apo-structure as an initial model. Model
AUGUST 5, 2011•VOLUME 286•NUMBER 31
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Crystal Structure of Uronate Dehydrogenase
TABLE 1
Data collection and refinement statistics
PDB, Protein Data Bank; r.m.s.d., root mean square deviation.
Apo-structure
KAu(CN)2
NADH and product
Y136A
Beamline
ID14-4
ID14-4
P6222
X12
ID23-1
3RFX
P6222
PDB code
3RFT
3RFV
Space group
P6222
P6222
Unit cell (Å)
a and b ϭ 164.7, c ϭ 174.8
a and b ϭ 164.2, c ϭ 174.5
1.9
a and b ϭ 165.4, c ϭ 173.6
a and b ϭ 165.5, c ϭ 174.1
Resolution (Å)
High resolution shell (Å)
1.9
2.1
1.9
1.95-1.90
8.2 (32.9)
100 (100)
5.7 (5.7)
18.3 (4.8)
15.1
1.95-1.90
2.15-2.10
8.0 (36.9)
99.8 (100)
11.0 (11.0)
27.3 (6.8)
15.7
1.95-1.90
7.1 (38.2)
97.6 (98.9)
11.0 (11.1)
23.3 (6.1)
15.8
R
merge (%)
6.9 (33.6)
Completeness (%)
Multiplicity
I/␴I
95.7 (87.0)
5.7 (3.8)
21.5 (4.3)
R-factor (%)
R
free (%)
17.2
18.9
18.0
No. of atoms
7066
7114
7118
Protein
6175
6132
6111
Water
876
771
896
NADH
132
81
Product lactone
Other
39
15 (SO4)
20.1
40 (PO4)
23.0
30 (PO4)
21.4
Mean B-factor (Å2)
NADH
24.7
27.4
Product lactone
r.m.s.d. bond length (Å)
r.m.s.d. bond angles
Ramachandran plot (%)
Most favored
Additionally allowed
35.1
0.007
0.007
1.091°
0.007
1.052°
1.052°
91.5
8.5
90.8
9.2
92.2
7.8
building was carried out with COOT, and the refinement was
performed with PHENIX. The validation of the models was
performed with PROCHECK (15). The data collection and
refinement statistics are summarized in Table 1.
Analysis of the crystal structure of AtUdh with the PISA
server (17) suggests that the enzyme forms a hexamer with a
total interface area of ϳ7450 Å2. Two monomers form a
tightly packed dimer (interface area of 1400 Å2), and there is
a 2-fold symmetry axis between these monomers. Helices ␣3
and ␣4 from both monomers are packed together to form a
four-helical bundle (Fig. 1B). Three of these dimers are then
more loosely packed together around a 3-fold symmetry axis,
perpendicular to the symmetry axes between the tight
dimers (Fig. 1, B and C). This packing results in a total of six
monomer-monomer interfaces of 540 Å2. AtUdh was also
found to be a hexamer in solution, as analyzed by size-exclu-
sion chromatography and dynamic light scattering (data not
shown).
Free Cysteines—AtUdh contains six free cysteine residues,
which do not form disulfide bridges, typical for a cytosolic
enzyme. Cys-65, Cys-166, and Cys-200 are embedded in the
hydrophobic environment in the protein interior. The side
chain of Cys-141 is on the protein surface but located on the
monomer-monomer interface. Cys-47 and Cys-50, located in
the ␤3-strand, extend to the protein outer surface and may be
theoretically capable of making intermolecular disulfide
bridges in an oxidative environment.
RESULTS
Structure Determination—The protein crystals of AtUdh
belong to the space group P6222. There are three AtUdh mono-
mers in the asymmetric unit, and the Matthews coefficient (16)
is 3.8 Å3/Da, which corresponds to an estimated solvent con-
tent of 68%. The apo-structure of AtUdh was solved by x-ray
crystallography at 1.9 Å resolution and phased by the SIRAS
method with a gold derivative. The final model consists of 813
residues and 876 water molecules. The apo-structure was
refined to an R-factor of 15% and a Rfree value of 17%. The root
mean square differences between the monomers are ϳ0.2 Å.
The complex structure with NADH and product was deter-
mined by soaking the crystals with a substrate, D-galacturonic
ϩ
acid, and a cofactor, NAD . The resolution of the structure is
2.1 Å, the R-factor is 15.7%, and the Rfree value is 18.9%. The
ϩ
Y136A mutant complexed with NAD was refined to 1.9 Å
with an R-factor of 16.1% and an Rfree value of 18.6%. The root
mean square differences between the molecules and models are
ϳ0.2 Å.
Nucleotide-binding Site—In the electron density maps of the
ternary complex of AtUdh, clear electron density was observed
at the expected dinucleotide-binding site, which allowed an
unambiguous fit of NADH with the electron density (Fig.
1D). All of the residues and solvent molecules that directly
interact with NADH were identified (Fig. 2B). The bound
NADH is located at the C-terminal edge of the ␤-sheet in an
extended conformation. The adenine ring is positioned in an
anti-conformation, the nicotinamide ring moiety is in a syn-
conformation, and both riboses of the NADH adopt an
C2-endo-conformation.
Overall Structure—The AtUdh monomer is a single-domain
protein consisting of seven ␤-strands and seven ␣-helices. The
structure of AtUdh contains a Rossmann fold motif with a par-
allel seven-stranded ␤-sheet (␤1–␤7) surrounded by five ␣-
helices (␣2, ␣3, and ␣4 on one side and ␣1 and ␣6 on the other
side). The cofactor is bound in a crevice at the C-terminal side
of the ␤-sheet between ␤1 and ␤4, in which the strand order is
reversed. Two shorter ␣-helices (␣5 and ␣7) are located away
from the main domain body and constitute parts of the sub-
strate-binding cleft. The secondary structure elements of
AtUdh are shown in Fig. 1A.
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Crystal Structure of Uronate Dehydrogenase
FIGURE 1. A, overall structure of the AtUdh monomer. B, hexameric structure of AtUdh. Two AtUdh units form a tight dimer (purple and red). A complete
hexamer contains two additional dimers (gray). C, AtUdh hexamer as in B but rotated by 90°. D, electron density map around NADH and the product
D-galactaro-1,5-lactone. E, electron density map around NADϩ in the Y136A structure. The electron density omit maps (Fo Ϫ Fc) are contoured at 2␴.
The adenine ring of NADH interacts with the enzyme via 174. The O1 carbonyl oxygen of the lactone is hydrogen-
hydrophobic interactions with Leu-35 and Leu-52 and a hydro- bonded to Tyr-136 and Ser-111, and the O2 hydroxyl is bonded
gen bond between the adenine amino group and the Asp-51 to the main-chain oxygen of Ser-75. The distance between the
carboxyl group. The 2Ј- and 3Ј-hydroxyl groups of the adenine reactive C1 of the carbohydrate and nicotinamide C4 is 3.4 Å.
ribose form hydrogen bonds with Asp-34. In addition, there is a As a result of this, the two molecules are very well placed for the
hydrogen bond between the 2Ј-hydroxyl group and Ser-36. The hydride transfer.
presence of Asp-34 explains especially well why AtUdh does
Active-site Mutation Y136A—A putative proton acceptor,
ϩ
not accept NADP as a cofactor, as the negatively charged Tyr-136 was substituted with alanine to trap the substrate or a
ϩ
phosphate at the 2Ј-position would repel a negatively charged reaction intermediate in the crystal in the presence of NAD
carboxyl group of Asp-34. The pyrophosphate moiety is hydro- and the substrate D-galacturonic acid. The activity of the puri-
gen-bonded to the main-chain atoms of Gln-14 and Leu-15 and fied Y136A mutant on D-galacturonic acid was ϳ0.1% of the
to the side chain of Ser-75. The 3Ј-hydroxyl group of the nico- activity of wild-type AtUdh. The consequences of the mutation
tinamide ribose is hydrogen-bonded to Lys-140, and the 2Ј-hy- were also clearly visible in the electron density map. Surpris-
droxyl group forms a hydrogen bond with Tyr-136. The nico- ingly, there was no clear electron density for the substrate car-
ϩ
tinamide ring has hydrophobic interactions with Ile-163, and bohydrate or for the nicotinamide residue of the NAD cofac-
the oxygen of the amide group forms a hydrogen bond with the tor. However, electron density was visible for the adenine and
ϩ
main-chain nitrogen of Cys-166.
both phosphates of NAD (Fig. 1E). The nicotinamide residue
Substrate Binding—The refinement of the AtUdh apo-struc- probably had multiple conformations because the electron den-
ture showed in the active site the electron density of a sulfate sity continues in several directions. Comparison of the mutant
ion, which was used in crystallization (Fig. 2A). The oxygen structure with the apo-structure and the ternary structure
atoms of the sulfate make hydrogen bonds with the side chains revealed an ϳ1.5-Å shift in the positions of Ile-74 and Ser-75
ϩ
of Asn-112, His-113, Ser-165, and Arg-174. The sulfate was toward the nicotinamide ribosyl ring of NAD . This may have
shown to inhibit AtUdh with an IC50 of ϳ40 mM. Consequently, restricted the binding of the nicotinamide end of the nucleotide
new crystallization conditions with ammonium phosphate and reduced enzymatic activity (Fig. 2D).
were used instead.
Reaction Product Analysis—Negative-ion electrospray ioni-
ϩ
The soaking of AtUdh crystals with NAD and D-galactu- zation MS was used to analyze end product(s) of the oxidation
ronic acid resulted in a crystal structure that showed an elec- reaction (supplemental Fig. S1). The mass spectrum of the
tron density ring in the active site close to the nicotinamide ring starting compound, D-galacturonic acid (194 Da), showed an
Ϫ
of the NADH. This density was interpreted as the reaction intense peak at m/z 193, corresponding to the [M Ϫ H] ion.
product D-galactaro-1,5-lactone (Fig. 1D). The lactone ring After hydrogen/deuterium exchange, the peak was observed at
stacks with the nicotinamide ring. The acid group of the lactone m/z 197 instead (data not shown), consistent with the exchange
occupies the same position as the sulfate ion in the AtUdh apo- of four hydrogens (accounting for four OH groups). The colli-
structure, and it is hydrogen-bonded to the side chain of Arg- sion-induced dissociation MS/MS spectrum of D-galacturonic
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Crystal Structure of Uronate Dehydrogenase
FIGURE 2. A–C, active-site residues and ligands in apo-AtUdh, in the NADH and product ternary complex, and in the Y136A mutant structure, respectively. D, all
three structures superimposed. NADH is in green, the product in cyan, and sulfate in yellow. E, superimposition of AtUdh (in purple) and S. venezuelae DesIV (in
gray) showing the binding of NADH and product for AtUdh (in cyan) and substrate for DesIV (gray). F, binding of the reaction product D-galactaro-1,5-lactone
showing the key distances (in Å) between the ligand (in cyan), cofactor (in green), and active-site residues (in purple).
acid showed a plethora of fragments. Two consecutive water stabilized ion at m/z 85. This is in line with the formation of a
losses (ions at m/z 175 and 157) were observed with the [M Ϫ 1,4-lactone ring. In the case of a 1,5-lactone (six-membered
Ϫ
H] ion. Alternatively, decarboxylation (elimination of CO2) ring), the first decarboxylation would result in the carbanion
was observed (m/z 149), which is highly characteristic for car- structure, similar to the ion at m/z 149, from which the ring
boxylic acid anions, followed by two water losses (m/z 131 and opening cannot directly occur without rearrangement (e.g.
113) or loss of H2CO (m/z 119) or CH2(OH)CHO (m/z 89).
hydride shift). Given the high abundance of the ion at m/z 85, it
The mass spectrum of the reaction product(s) showed an is evident that the more stable reaction product is D-galactaro-
intense peak at m/z 191 as well as a few other peaks that were 1,4-lactone. However, the initial reaction product is 1,5-lac-
identified as the impurities of the enzyme solution (supplemen- tone, as evidenced by crystallographic results. No other reac-
tal Fig. S1, marked with asterisks). After hydrogen/deuterium tion products were detected.
exchange, the peak was observed at m/z 194 instead. These
DISCUSSION
results are consistent with the formation of D-galactarolactone
(192 Da), having three OH groups in the structure. The MS/MS
Substrate Specificity—The ternary crystal structure clearly
ϩ
spectrum of the reaction product showed a very intense peak at suggests that the binding of the NAD cofactor is a prerequisite
m/z 85 and less abundant fragment ions at m/z 173, 147, 129, for substrate binding because it participates in the formation of
and 111. This can be explained by decarboxylation (m/z 147), a ligand-binding site (Fig. 2F). The structure also shows that
followed by a water loss (m/z 129) and a second decarboxylation AtUdh is able to bind substrates in a pyranose form, i.e. as a
(m/z 85). Inspection of the putative ion structures for the cyclic six-membered ring. The specific activity of AtUdh has
deprotonated D-galacturonic acid indicates that the fragmenta- been studied recently by Yoon et al. (7) and more extensively by
tion proceeds without ring opening (forming a stable cyclic car- Boer et al. (6) These results are in good agreement with the
banion structure, from which water eliminations can occur). In crystal structures. AtUdh accepts both D-glucuronic acid and
contrast, for the deprotonated D-galactarolactone (five-mem- D-galacturonic acid as substrates. These sugar acids have a dif-
bered ring), the first decarboxylation results in the immediate ference in the position of O4 hydroxyl group. According to the
ring opening, followed by water elimination and a second complex structure, the axial O4 hydroxyl group of D-galactu-
decarboxylation. This results in a fully conjugated, resonance- ronic acid is toward a solvent and does not form any hydrogen
27298 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of Uronate Dehydrogenase
bonds, and there would be space for the O4 hydroxyl group in
an equatorial position (Fig. 2F). D-Galactose, D-glucose, D-ga-
lactonic acid, and D-gluconic acid are not substrates for AtUdh
(6). All of these compounds lack the carboxyl group at C6,
which suggests that interaction of the carboxyl group with Arg-
174 is essential for substrate binding.
Structure Comparison with Other SDRs—The SDR family is
one of the largest enzyme families (18). However, there are only
a few distantly related structures in the Protein Data Bank. Only
dTDP-glucose 4,5-dehydratase (DesIV) from Streptomyces ven-
ezuelae shows some amino acid sequence identity (18%) to
AtUdh (19). DesIV catalyzes a much more complicated reac-
tion by oxidizing the 4Ј-hydroxyl group and by removing the
6Ј-hydroxyl group of the dUTP-glucose substrate. DesIV is a
ϩ
55-residue larger protein than AtUdh. The NAD -binding sec-
tion is similar, but there are large differences in the structure,
especially in the C-terminal region. The wild-type DesIV com-
ϩ
plex structure with NAD and substrate does not show the
electron density for glucose, whereas the inactive double
mutant (D128N/E129Q) does. Superimposition of the cofac-
tor- and substrate-binding sites of AtUdh and DesIV is shown
in Fig. 2E. Despite large differences in the overall protein struc-
tures, especially in the loops, the binding modes for the cofactor
and carbohydrates resemble each other.
Mechanism—The reaction catalyzed by AtUdh has been
studied by NMR (6). The ␤-anomeric form of D-galacturonic
acid was first consumed faster than the ␣-anomer. This is
explained nicely by the ternary complex structure, which would
suggest that the O1 hydroxyl group of ␣-D-galacturonic acid
ϩ
would clash with the nicotinamide ring of NAD . Conse-
quently, AtUdh would accept only ␤-D-galacturonic acid as a
substrate. This is further formed in solution from ␣-D-galactu-
ronic acid by mutarotation.
NMR analysis also suggested that the reaction product would
be D-galactaro-1,4-lactone (five-membered ring) and not D-ga-
lactaro-1,5-lactone (six-membered ring), as observed in the ter-
nary complex structure. There was also a small amount of
meso-galactarate in solution. This is in line with the observa-
tions made by electrospray ionization MS in this study. It is
reasonable to assume that the reaction product D-galactaro-
1,5-lactone rearranges non-enzymatically to D-galactaro-1,4-
lactone, possibly via an open-chain meso-galactaric acid form
(Fig. 3). Similar non-enzymatic 1,4-lactone formation has been
observed previously for D-galactose dehydrogenases (20).
Because the rearrangement is an equilibrium reaction, a very
small amount of D-galactaro-1,5-lactone exists in solution and
FIGURE 3. Proposed reaction mechanism for AtUdh. The locations of steps
in water solution and in enzyme are indicated. In solution, the substrate D-ga-
lacturonic acid exists mainly in the equilibrium of ␣- and ␤-pyranosic forms of
the D-galacturonic acid (6), but only the ␤-anomeric form binds to the active
site of the enzyme. In the enzymatic reaction, the removal of hydride by
NADϩ creates a resonance-stabilized cationic intermediate. The subsequent
removal of a proton by Tyr-136 creates the reaction product D-galactaro-1,5-
lactone, which is rearranged to the more stable D-galactaro-1,4-lactone in
solution.
explains why this form was observed in the ternary complex as a non-concerted or a stepwise reaction. Therefore, it is
crystal structure. important to consider which is the first step. Proton donation
The catalytic mechanism of the dehydrogenase activity of the from the O1 hydroxyl group to the catalytic tyrosine would
SDR family enzymes has been extensively discussed in light of create a negatively charged oxyanion, which would have high
structural, enzymological, and mutational data. It has been sug- energy (verified normally by high pKa values of hydroxyl
gested that there would be a catalytic triad (Ser, Tyr, and Lys) or groups). This kind of initiation has been proposed recently by
even a catalytic tetrad (Ser, Tyr, Leu, and Asn) and a proton Yasutake et al. (23). On the other hand, the hydride shift from
ϩ
relay system for SDRs (21). A classical proposed reaction mech- C1 to NAD would lead to the resonance-stabilized cationic
anism for this enzyme family includes a transition state in intermediate. This intermediate would eagerly donate a proton
which several bonds form and break at the same time (22). to the neighboring group, in this case to Tyr-136, which in
However, to describe the reaction as a “single-barrier” process AtUdh is an unusually short distance (2.5 Å) from the carbonyl
might be an oversimplification. Rather, it could be considered oxygen of the product (Fig. 2F). AtUdh could efficiently cata-
AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27299

Crystal Structure of Uronate Dehydrogenase
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Acknowledgments—We thank Armi Boman, Arja Kiema, and Outi
Liehunen (VTT Technical Research Centre of Finland) and Ritva
Romppanen (University of Eastern Finland) for technical assistance.
27300 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Protein Structure and Folding:
Crystal Structure of Uronate
Dehydrogenase from Agrobacterium
tumefaciens
Tarja Parkkinen, Harry Boer, Janne Jänis,
Martina Andberg, Merja Penttilä, Anu
Koivula and Juha Rouvinen
J. Biol. Chem. 2011, 286:27294-27300.
doi: 10.1074/jbc.M111.254854 originally published online June 15, 2011
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This article cites 23 references, 4 of which can be accessed free at
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