Structural and functional insights into (S)-ureidoglycine aminohydrolase, keyenzyme of purine catabolism in Arabidopsis thaliana

Article abstract of DOI:10.1074/jbc.M111.331819

The ureide pathway has recently been identified as the metabolic route of purine catabolism in plants and some bacteria. In this pathway, uric acid, which is a major product of the early stage of purine catabolism, is degraded into glyoxylate and ammonia via stepwise reactions of seven different enzymes. Therefore, the pathway has a possible physiological role in mobilization of purine ring nitrogen for further assimilation. (S)-Ureidoglycine aminohydrolase enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, we report a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH). The crystal structure of AtUGlyAH in the ligand-free form shows a monomer structure in the bicupin fold of the β-barrel and an octameric functional unit as well as a Mn²⁺ ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn²⁺ ion acts as a molecular anchor to bind (S)-ureidoglycine, and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn²⁺ ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism.

Full text of DOI:10.1074/jbc.M111.331819

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 22, pp. 18796–18805, May 25, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Structural and Functional Insights into (S)-Ureidoglycine
Aminohydrolase, Key Enzyme of Purine Catabolism in
□
S
Arabidopsis thaliana*
Received for publication, December 7, 2011, and in revised form, March 13, 2012 Published, JBC Papers in Press, April 5, 2012, DOI 10.1074/jbc.M111.331819
Inchul Shin‡, Riccardo Percudani§, and Sangkee Rhee‡¶1
From the ‡Department of Agricultural Biotechnology and the ¶Center for Fungal Pathogenesis, Seoul National University,
Seoul 151-921, Korea and the §Department of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy
Background: (S)-Ureidoglycine aminohydrolase plays a key role in purine catabolism of plants and some bacteria.
Results: Substrate (S)-ureidoglycine is bound to the Mn2ϩ ion at the active site of homo-octameric enzyme.
Conclusion: The binding mode of (S)-ureidoglycine dictates the enantioselectivity of the reaction.
Significance: (S)-Ureidoglycine aminohydrolase liberates one of purine ring nitrogen as ammonia, which could be utilized for
further nitrogen assimilation.
The ureide pathway has recently been identified as the meta-
bolic route of purine catabolism in plants and some bacteria. In
this pathway, uric acid, which is a major product of the early
stage of purine catabolism, is degraded into glyoxylate and
ammonia via stepwise reactions of seven different enzymes.
Therefore, the pathway has a possible physiological role in
mobilization of purine ring nitrogen for further assimilation.
(S)-Ureidoglycine aminohydrolase enzyme converts (S)-ureido-
glycine into (S)-ureidoglycolate and ammonia, providing the
final substrate to the pathway. Here, we report a structural and
functional analysis of this enzyme from Arabidopsis thaliana
(AtUGlyAH). The crystal structure of AtUGlyAH in the ligand-
free form shows a monomer structure in the bicupin fold of the
␤-barrel and an octameric functional unit as well as a Mn2؉ ion
binding site. The structure of AtUGlyAH in complex with (S)-
ureidoglycine revealed that the Mn2؉ ion acts as a molecular
anchor to bind (S)-ureidoglycine, and its binding mode dictates
the enantioselectivity of the reaction. Further kinetic analysis
characterized the functional roles of the active site residues,
including the Mn2؉ ion binding site and residues in the vicinity
of (S)-ureidoglycine. These analyses provide molecular insights
into the structure of the enzyme and its possible catalytic
mechanism.
and catabolism of nitrogen-rich metabolites (1). Of these, nitro-
gen assimilation is a universal, indispensible process common
to all organisms, as identified in de novo biosynthesis of purines
and pyrimidines (2, 3). In contrast, both nitrogen uptake and
degradation of nitrogenous compounds manifest in distinct
metabolic pathways in different organisms (4).
Recently, the unique nitrogen utilization features of the
pyrimidine and purine catabolic pathways have been revealed.
For example, two oxidative and reductive pyrimidine degrada-
tion pathways have been characterized in Escherichia coli (5).
An additional Rut pathway was also recently identified in this
bacterium (6, 7). In contrast, an oxidative purine degradation
pathway is conserved in most organisms, the primary product
of which is uric acid (1). Although uric acid has a high content of
nitrogen, with a nitrogen-to-carbon ratio of 0.8 (supplemental
Fig. S1), humans and most mammals are unable to utilize the
ring nitrogen in uric acid due to a lack of the necessary catabolic
pathways. However, there exists a recently elucidated uric acid
degradation pathway that could remobilize the ring nitrogen
atoms for further assimilation. This catabolic pathway in purine
metabolism, called the ureide pathway, has been characterized
in Arabidopsis thaliana, and the presence of genes for the path-
way were also identified in all plants that have been sequenced
to date as well as in some bacteria and fungi (8–10). These two
independent catabolic pathways (the Rut pathway for pyrimi-
dines and the ureide pathway for purines) do not show any
genetic or biochemical resemblance but exhibit common fea-
tures for producing ammonia as a reaction byproduct. There-
fore, the possible physiological role of these degradation path-
ways has been suggested for nitrogen mobilization from
nitrogen-rich metabolites in their respective organisms (4, 7, 9,
10).
Nitrogen metabolism is a biological process present in a wide
variety of living organisms, and it is crucial in cell physiology
because it supplies an essential nutrient. Nitrogen metabolism
includes metabolic routes for nitrogen uptake and assimilation
* This work was supported by grants from the Next-Generation BioGreen 21
Program (Plant Molecular Breeding Center no. PJ008059), Rural Develop-
ment Administration, and from National Research Foundation (R11-2008-
062-01002-0 and 2010-0025883) funded by the Ministry of Education, Sci-
ence, and Technology, Republic of Korea.
The ureide pathway in A. thaliana is composed of seven
enzymes and has been studied extensively via biochemical and
structural investigations (9–17). The pathway begins with uric
□S
This article contains supplemental Tables S1–S4 and Figs. S1–S7.
The atomic coordinates and structure factors (codes 4E2Q and 4E2S) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural acid and produces a stereospecific (S)-allantoin through a
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
three-step enzyme reaction occurring in peroxisome (supple-
mental Fig. S1) (8, 18, 19). Subsequently, (S)-allantoin is trans-
ported into the endoplasmic reticulum and is subject to further
1 To whom correspondence should be addressed: Dept. of Agricultural Bio-
technology, Seoul National University, Seoul 151-921, Korea. Tel.: 82-2-
880-4647; Fax: 82-2-873-3112; E-mail: srheesnu@snu.ac.kr.
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
stepwise reactions of four other enzymes, resulting in the pro-
duction of 1 eq of glyoxylate and 4 eq of ammonia molecules
from 1 molecule of (S)-allantoin (9, 10). In contrast, bacterial
enzymes catalyze the conversion of (S)-ureidoglycolate, which
is the last substrate to the pathway in A. thaliana, into either
oxalurate or glyoxylate depending on oxygen and/or nitrogen
availability (supplemental Fig. S1).
SCHEME 1. Conversion of (S)-ureidoglycine into (S)-ureidoglycolate by
UGlyAH. The full ureide pathway is depicted in supplemental Fig. S1.
(S)-Ureidoglycolate is thus the end product of the ureide
pathway common to A. thaliana as well as some bacteria in minimal medium supplemented with seleno-L-methionine.
and fungi. The enzyme (S)-ureidoglycine aminohydrolase When the optical density of the culture medium reached 0.7 at
(UGlyAH)2 (EC 3.5.3.-) catalyzes the conversion of (S)-ureido- 600 nm, the recombinant protein was expressed for 18 h at
glycine into (S)-ureidoglycolate in an enantioselective manner 20 °C with the addition of 0.5 mM isopropyl-1-thio-␤-D-galac-
using a Mn2ϩ ion as a co-factor and releases ammonia (see topyranoside. The cells were harvested and sonicated in buffer
Scheme 1) (9). In this study we carried out structural and func- A (50 mM NaH2PO4, pH 7.4, and 200 mM NaCl), and the super-
tional analyses of UGlyAH from A. thaliana (AtUGlyAH). In natant was obtained by centrifugation at 30,000 ϫ g for 1 h at
particular, the structure of AtUGlyAH in the ligand-free form 4 °C. The enzyme was purified using an immobilized metal
and as a complex with (S)-ureidoglycine substrate was deter- affinity column (GE Healthcare) that had been equilibrated
mined, thus providing molecular insights into the structure of with buffer A and was eluted with buffer B (buffer A containing
this enzyme and its mechanism.
500 mM imidazole). After the pooled elution fractions were dia-
lyzed against buffer C (50 mM NaH2PO4, pH 7.4, and 100 mM
NaCl), the N-terminal His tag was removed by treatment of
tobacco etch virus protease for 12 h at 4 °C. AtUGlyAH in the
absence of a His tag was further purified by immobilized metal
affinity chromatography followed by gel filtration chromatog-
raphy using Superdex-200 (GE Healthcare) with buffer D (50
mM HEPES, pH 7.4, and 100 mM NaCl). The protein was con-
centrated to ϳ10 mg/ml, and 1 mM MnCl2 and 5 mM DTT were
added before crystallization.
EXPERIMENTAL PROCEDURES
Construct—The UGlyAH gene from A. thaliana (Gen-
BankTM accession number GQ303359) was modified and used
as a template for PCR. The modifications were performed using
the QuikChange method (Agilent) to introduce a silent muta-
tion at the sequences for NdeI and XhoI sites residing in the
internal region of the AtUGlyAH gene. The N-terminal trun-
cated variants of AtUGlyAH, from which the signal peptide
(residues 1–20) and an additional N-terminal region had been
removed, were amplified using a pair of sequence-specific
primers (supplemental Table S1). The resulting PCR products
were introduced into the NdeI and XhoI sites of a modified
pET-28a vector (Merck) containing a tobacco etch virus prote-
ase cleavage site at the junction between a His5 tag and a mul-
tiple cloning site. For the functional assay, all AtUGlyAH
mutants were also prepared using the QuikChange method
with mutagenic primers (supplemental Table S1).
Crystallization using seleno-L-methionine-AtUGlyAH was
initially carried out by the sitting-drop vapor-diffusion method
at 295 K. Of the various constructs, AtUGlyAH (⌬35), from
which the N-terminal 35 residues had been removed, produced
crystals suitable for further structural determination under two
different conditions: crystal A grown under 0.1 M phosphate
citrate, pH 4.2, 10% (w/v) PEG 3000, 0.2 M NaCl and crystal B
grown under 0.1 M HEPES, pH 7.5, 7% (w/v) PEG 8000, 8% (v/v)
ethylene glycol. These conditions were further optimized using
the hanging-drop vapor-diffusion method at 295 K.
We produced (S)-ureidoglycine, and the enzyme activity of
AtUGlyAH was assayed using E. coli (S)-ureidoglycolate dehy-
drogenase as a coupled enzyme (supplemental Fig. S1). There-
fore, expression vectors were constructed as described above
for genes encoding the enzymes necessary for producing (S)-
ureidoglycine from a racemic allantoin mixture and also for the
gene encoding (S)-ureidoglycolate dehydrogenase. Those were
as follows: allantoinase from E. coli (14), allantoate amidohy-
drolase from A. thaliana (gene access no. NM_11826) and
E. coli strain K-12 substrain DH10b (EcAAH; gene access no.
NC_010473), and (S)-ureidoglycolate dehydrogenase (gene
access no. NC_010473) from E. coli strain K-12 substrain
DH10b.
Data Collection and Structure Determination—X-ray dif-
fraction data were collected on beamline 4A and 6C at the
Pohang Accelerator Laboratory, Korea. Both crystal A and B
were cryoprotected by soaking each in crystallization solution
containing additional 20–25% (v/v) glycerol and then flash-
frozen in a liquid nitrogen stream at 100 K. Initially, single-
wavelength anomalous diffraction data were collected to 3.0 Å
resolution on crystal A at a wavelength of 0.97969 Å, corre-
sponding to an absorption peak of selenium, and 2.5 Å resolu-
tion on crystal B. Subsequently, data for the enzyme-substrate
binary complex using crystal B were collected to 2.6 Å resolu-
tion. Formation of the binary complex was achieved by a soak-
ing experiment in which crystal B was soaked for 60 s in a solu-
tion containing crystallization solution, 20% glycerol, and 3 mM
(S)-ureidoglycine. To prepare (S)-ureidoglycine, 15 mM allan-
toate (Sigma) was incubated with allantoate amidohydrolase
from A. thaliana (2.5 ␮g/␮l; 49.2 ␮M) and 1 mM MnCl2 for 2
min (supplemental Fig. S2A). The reaction product was then
diluted with crystallization solution containing 20% glycerol.
The collected data were processed using HKL2000 (20). Crystal
Protein Expression, Purification, and Crystallization—For
structural studies, seleno-L-methionine-substituted, N-termi-
nal His-tagged AtUGlyAH was expressed in E. coli B834 (DE3)
methionine auxotroph cells (Merck) that were grown at 37 °C
2 The abbreviations used are: UGlyAH, (S)-ureidoglycine aminohydrolase;
AtUGlyAH, UGlyAH from A. thaliana; EcAAH, allantoate amidohydrolase
from E. coli; r.m.s.d., root mean-square deviation.
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
A belongs to a P3221 space group with a tetramer in an asym- NADPH (9, 30). This method was performed for various con-
metric unit, whereas crystal B belongs to a P21 space group with
16 monomers in an asymmetric unit (supplemental Table S2).
Initially, the structure of crystal A was determined using the
SOLVE (21) and RESOLVE (22, 23) programs for phasing and
density modification, respectively. Even with 3.0 Å resolution
data, the initial electron density map was sufficient to trace
most of the residues in each monomer. Molecular model build-
ing and refinement was performed using COOT (24) and CNS
(25), respectively, and the PHENIX program (26) was used for
molecular replacement. Of the four monomers in the crystal A
structure, one was manually modeled and then used as a search
model of molecular replacement for locating three other sub-
units. Further refinement of the tetrameric structure in crystal
A was carried out, and Rwork and Rfree values were 23.5 and
30.6%, respectively. Subsequently, higher resolution data were
available using a crystal B, and we conducted molecular
replacement using the refined monomer in crystal A as a search
structure. In the 2.5 Å resolution structure of crystal B, a total of
12 monomers of the 16 in the asymmetric unit were initially
found. At this stage, one particular monomer was manually
modeled and again used as a search model for further molecular
replacement. Eventually, all 16 monomers were found and
automatically built in PHENIX. These 16 monomers were then
manually inspected and refined to Rwork and Rfree values of 22.4
and 28.6%, respectively. For the AtUGlyAH in complex with
(S)-ureidoglycine, the refined structure of the ligand-free form
was used as a starting model for refinement. During refinement,
we noticed an electron density corresponding to (S)-ureidogly-
cine in the metal binding site, and its model was built and
included in the structure for further refinement. The stereo-
chemistry of the refined models was evaluated using a program
MolProbity (27). Details of data collection and refinement are
in supplemental Table S2. Structural analyses were carried out
using programs in the CCP4 suite (28). The figures presented in
this study were prepared using PyMol (29).
trol experiments and used to verify the substrate specificity of
each purified enzyme (supplemental Fig. S2, A and B). The reac-
tion mixture (2 ml) contained 0.2 M Tris-HCl buffer, pH 8.5, 0.3
mM NADPH, 2.5 mM ␣-ketoglutarate, 10 units of glutamate
dehydrogenase from Proteus sp. (Sigma), 100 ␮M MnCl2, and
800 ␮g of EcAAH (8.3 ␮M). The reaction was then initiated by
the addition of 75 ␮M allantoate (Sigma) or generated from
allantoin (see below) followed by the addition of AtUGlyAH
(0.0581–0.232 ␮M). The reaction progress curve proved that
the enzymes used in this assay were functionally active and
exhibited high substrate specificity.
Measurements of the steady-state kinetic parameters of
AtUGlyAH WT and mutant enzymes were performed using the
second method, mainly because the first protocol was imprac-
tical at high substrate concentrations. Specifically, in our pro-
tocol, formation of (S)-ureidoglycolate by AtUGlyAH was
monitored in a coupled assay with (S)-ureidoglycolate dehydro-
genase in which the (S)-ureidoglycolate produced is subse-
quently converted into oxalurate, with concurrent reduction of
ϩ
NAD into NADH accompanying an increase in absorbance at
340 nm. We verified the following requirements for reliable
kinetic measurements; under the experimental conditions out-
lined below, the increase in absorbance at 340 nm was depen-
dent only on the concentrations of (S)-ureidoglycine and
AtUGlyAH but not of other enzymes (supplemental Fig. S2C),
and allantoin was completely converted into (S)-ureidoglycine
(supplemental Fig. S2B). Initially, a 50 mM allantoin racemic
mixture (i.e. 25 mM (S)-allantoin) was dissolved in water at
37 °C for 1 h and cooled to 30 °C and then diluted with enzyme
reaction buffer (50 mM HEPES, pH 7.5) to a final concentration
of 20 mM (S)-allantoin. The reaction was initiated by the addi-
tion of allantoinase (0.525 mg/ml; 10 ␮M) at 30 °C for 1 h to
produce 20 mM allantoate. This allantoate solution was stored
at Ϫ70 °C and used without further purification. The assay mix-
ture solution (2 ml) contained 50 mM HEPES buffer, pH 7.5, 100
␮M MnCl2, (S)-ureidoglycolate dehydrogenase (0.2 mg/ml; 5.0
Enzyme Assay—For functional analysis, all enzymes used in
this study were prepared as described above, with the exception
of allantoinase. For expression, E. coli BL21 (DE3) (Merck) har-
boring the plasmid of interest was cultured in Luria-Bertani
medium. Subsequently, the N-terminal His-tagged protein was
purified by immobilized metal affinity chromatography and
dialyzed against buffer (50 mM Tris-HCl, pH 7.4, and 200 mM
NaCl). For allantoinase, functionally active enzyme was
expressed and purified as described previously (14).
ϩ
␮M), 3 mM NAD , and allantoate at the concentrations indi-
cated (0.2–16 mM). After incubation for 60 s, further conver-
sion of allantoate into (S)-ureidoglycine was catalyzed by the
addition of EcAAH (0.4 mg/ml; 8.3 ␮M), and (S)-ureidoglyco-
late formation was subsequently initiated by the addition of
AtUGlyAH (3.9 nM-0.15 ␮M) (supplemental Fig. S2C). Be-
cause(S)-ureidoglycine is chemically labile, the time period
required for complete conversion of allantoate to (S)-ureidogly-
cine was determined experimentally by measuring the initial
velocity of AtUGlyAH as a function of incubation time. Typi-
cally, 60 s of incubation was the optimal time for complete
formation of (S)-ureidoglycine under our experimental condi-
tions. Shorter or longer incubations caused a lower velocity,
which indicates that either the conversion was incomplete, or
chemically labile (S)-ureidoglycine underwent spontaneous
degradation, respectively. The initial velocity was determined
by measuring the linear increase in absorbance at 340 nm for
the first 20 or 30 s and was calculated as the NADH concentra-
Enzyme assays were performed at 30 °C using a UV-visible
spectrophotometer (Jasco V-560) equipped with a cuvette
holder and connected to a temperature-controlling water cir-
culator. We employed two different assay protocols; one for
monitoring the stepwise release of ammonia during the forma-
tion of (S)-ureidoglycolate and the other for measuring the ini-
tial velocity of the formation of (S)-ureidoglycolate in a coupled
reaction with an NAD-dependent (S)-ureidoglycolate dehydro-
genase reaction.
In the first method the release of ammonia was monitored in
a coupled assay with an NADPH-dependent glutamate dehy-
drogenase in the presence of ␣-ketoglutarate by measuring the
tion produced per min, with a molar extinction coefficient of
decrease in absorbance at 340 nm due to the oxidation of 6220 MϪ1cm
at 340 nm. The Km and Vmax values were
Ϫ1
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
FIGURE 1. Sequence alignment and overall structure of AtUGlyAH. A, the amino acid sequences of AtUGlyAH, including the signal peptide (residues 1–20)
(9), are compared with those of the E. coli ortholog and structural homologs from other bacteria, all with known structures in the absence of their functional
roles. Structures of these proteins were previously determined by Structural Genomics Centers: an E. coli ortholog (PDB ID 1RC6; resolution, 2.6 Å; Z-score, 29.6;
r.m.s.d., 2.1 Å; sequence identities, 28%) and structural homologs from Deinococcus radiodurans R1 (PDB ID 1SFN; 2.46 Å; 34.8; 1.4 Å; 40%), Enterococcus faecalis
V583 (PDB ID 1SEF; 2.05 Å; 32.3; 1.7 Å; 31%), and Pseudomonas aeruginosa (PDB ID 1SQ4; 2.7 Å; 28.3; 2.5 Å; PDB ID 26%). Highly conserved residues are shown
in red and boxed in blue. Strictly conserved residues are shown on a red background. The secondary structural elements defined in a ligand-free form are shown
for the corresponding AtUGlyAH sequences, with the N and C domains in red and blue, respectively. Black asterisks and triangles represent residues involved in
the Mn2ϩ coordination shell and the (S)-ureidoglycine binding site, respectively. Residues involved in the interactions between monomers within the layer are
indicated by blue rectangles; open and closed rectangles represent residues from different monomers. Cyan circles indicate residues involved in inter-layer
interactions between monomers located vertically, and magenta circles indicate those located diagonally across the layers. This figure was prepared using
ESPript (35). B, shown is the monomer structure, with an orientation identical to that of the monomer in gray in the left panel of C. The presence of a Mn2ϩ ion
is indicated by a black circle. Two residues, Thr-138 and Ser-139, are disordered in the loop between ␤6 and ␤7. C, shown is a surface representation of an
octamer in two orientations. A side view, with each monomer in a different color, is presented in the left panel, whereas the right panel shows a top view of the
layer.
obtained using SigmaPlot, and kcat values were computed by centrations were about 0.11ϳ0.14 mM, and the metal content of
dividing Vmax by the AtUGlyAH concentration used.
the enzyme was determined by inductively coupled plasma-
Metal Analysis—For metal analysis, the WT enzyme and atomic emission spectrophotometer.
E235Q and E235A mutants were expressed and purified as
RESULTS
described in “Enzyme Assay” above. Specifically, the N-termi-
nal His-tagged protein was purified by immobilized metal affin-
Molecular Architecture of Octameric AtUGlyAH in Ligand-
ity chromatography, and the His tag was subsequently removed free Form—The crystal structure of AtUGlyAH was determined
by treatment of tobacco etch virus protease. The His-tag free using AtUGlyAH ⌬35, which was missing 35 residues from the
enzyme was then incubated in the presence of 5 mM EDTA for N terminus including a cleavable signal peptide (Fig. 1A and
1 h at 4 °C and was purified by gel filtration chromatography. supplemental Table S2). Two octamers were characterized with
After the addition of 1 mM MnCl2, the resulting enzyme solu- no non-crystallographic symmetry in an asymmetric unit of the
tion was again subjected to gel filtration chromatography with P21 space group. The apparent molecular mass of AtUGlyAH
buffer (50 mM HEPES, pH 7.4, and 100 mM NaCl). Protein con- (30.2 kDa monomer) was estimated by size-exclusion chromatog-
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
FIGURE 2. Interactions of monomers within and between layers. Color code and orientation for each monomer in these figures are identical to those in the
left panel of Fig. 1C, and residues involved in these interactions are indicated with symbols as in Fig. 1A. A stereo view of these figures is shown in supplemental
Fig. S3. A, interactionsoftwoneighboringmonomerswithinthelayerareshown. B, twomonomerslocatedverticallyindifferentlayersarepresented. Residues,
such as Phe-230, Ile-251, Trp-258, Trp-276, Ala-278, Ala-279, and Leu-280 and equivalent residues from another monomer are clustered into this region to
mediate hydrophobic interactions as well as hydrogen bonding between the main chain Gly-228 and Gly-281*. C, interactions occurring between two
monomers located diagonally across the layers are shown. In particular, Lys-43, Thr-48, and Gln-249 participate in hydrogen bonding with Asp-56*, whereas
His-53 in the middle of the protruding region mediates a stacking interaction with the equivalent His-53* of a different monomer.
raphy to be more than 200 kDa, suggesting that the functional unit
of AtUGlyAH is likely an octamer.
Interactions between Monomers in Intra- and Inter-layers—
Within each layer, monomeric AtUGlyAH is packed against its
The crystal structure of monomeric AtUGlyAH, which con- neighbors, whose positions differ by 90° of rotation via hydro-
tains the ordered residues Pro-39 to Leu-298, is composed of 19 phobic interactions. These interactions are asymmetrical in
␤-strands and 4 short 310-helices (Fig. 1A). Its structure is that distinct regions from neighboring monomers participate
divided into two structurally similar ␤-barrel domains, called N in the packing. Specifically, residues in the loop regions of the
and C domains, with an N-terminal segment protruding from protruding N-terminal segment and those connecting ␤2 and
the rest of the structure (Fig. 1B). Each domain represents a ␤3 and connecting ␤3 and ␤4 mediate largely hydrophobic
typical cupin fold of a ␤-barrel, in which two layers of ␤-sheet interactions with other loop residues, mainly in the C domain
are packed in a parallel manner with a funnel-like structure ␤13*-␤14* and ␤17*-␤18* of the adjacent monomer (hereafter,
(31). Specifically, the N domain includes ␤3 to ␤11. Six strands an asterisk denotes an element or a residue of a neighboring
are arranged in the spatial order ␤3, ␤4, ␤10, ␤5, ␤8, and ␤11, monomer) (Fig. 2A and supplemental Fig. S3A). In addition, the
with an anti-parallel orientation. They form a central face of a C-terminal residues Asn-296* to Leu-298* are plugged into the
␤-barrel fold, whereas the outer face of the fold consists of ␤9, concave surface formed by the residues in the vicinity of the N
␤6, and ␤7, with their orientations parallel to the central domains ␤3 and ␤4. Several hydrogen bonds were character-
␤-sheet. The C domain also exhibits a similar architecture in ized in this packing within a layer (supplemental Fig. S3A). A
which N-terminal anti-parallel ␤1 and ␤2 strands form part of 4-fold arrangement of monomers in each layer generates four
the central anti-parallel ␤-sheet that comprises ␤1, ␤2, ␤17, independent but structurally identical interfaces; each interface
␤14, ␤19, ␤13, and ␤12 in a spatial order. Three strands, ␤16, exhibits a buried surface area of 1069 Å2 corresponding to
␤15, and ␤18, line along the outer surface of the ␤-barrel fold. ϳ8.8% of the surface area of the monomer.
Therefore, the AtUGlyAH monomer is a bicupin molecule and
More extensive, diverse packings were identified in the inter-
is stabilized by packing the central ␤-sheets from each domain actions between the two layers, with a total buried surface area
in a parallel manner (Fig. 1B). These interactions result in 2830 of 4677 Å2. The inter-layer interactions are symmetrical, in
Å2 of buried surface area and a root mean square deviation which structurally and sequentially identical residues are
(r.m.s.d.) of 1.9 Å for the structurally equivalent 91 C␣ atoms involved in the packing of monomers in different layers. Two
between the 2 cupin folds.
types of interactions are observed (Fig. 1C); one is between
The AtUGlyAH functional unit is assembled from two layers monomers located vertically in different layers, and the other is
of tetramers, each layer with a dorsal and a ventral surface (Fig. between monomers located diagonally across the layers. Spe-
1C). Within a layer, four monomers are related by a non-crys- cifically, residues in the outer surface of the C domain cupin
tallographic four-fold symmetry along the vertical axis running fold, including ␤16, ␤15, and ␤18, are major structural elements
through the center of the octamer, as shown by the top view of in stabilizing monomers orientated vertically in different layers
a dorsal surface. These four monomers are structurally identi- (Fig. 2B and supplemental Fig. S3B). In the other type of inter-
cal within 0.34–0.42 Å r.m.s.d. for 258 C␣ atoms. The two action, the protruding N-terminal region containing Pro-39 to
layers are stabilized by interacting through the ventral surface Ser-63 as well as residues on the outer surface of the C domain
but are offset from the vertical axis by ϳ22.5° of rotation (Fig. cupin fold in the vicinity of ␤16 and ␤15 are packed in a 2-fold
1C). Monomers in different layers are related by a 2-fold sym- symmetrical manner against the equivalent elements of a
metry along the two horizontal axes, which are orthogonal to monomer in a different layer (Fig. 2C and supplemental Fig.
each other. Therefore, a 422-symmetry along a vertical axis and S3C). Overall, these interactions serve as key structural features
two horizontal axes, respectively, fully describes the molecular in stabilizing the two layers, in particular by inserting the pro-
architecture of octameric AtUGlyAH using a monomer as a truding N-terminal segment into the concave surface of
protomer.
another monomer.
18800 JOURNAL OF BIOLOGICAL CHEMISTRY
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
our crystallization condition, the metal ion was identified as
Mn2ϩ ion by inductively coupled plasma-atomic emission
spectroscopy (supplemental Table S4). It is noticeable that
two water molecules ligating with the Mn2ϩ ion are near the
entrance, and therefore, their locations likely serve as the bind-
ing site for the incoming (S)-ureidoglycine substrate molecule.
Binding Mode of (S)-Ureidoglycine in Binary Complex—The
structure of AtUGlyAH in complex with (S)-ureidoglycine was
determined in a soaking experiment. We produced (S)-ureido-
FIGURE 3. Active site in the C domain cupin fold of the ligand-free form
glycine, which is commercially unavailable, by an enzyme reac-
tion using allantoate amidohydrolase with allantoate as the sub-
strate ( supplemental Figs. S1 and S2A ). By using the x-ray data
from a crystal of AtUGlyAH soaked with (S)-ureidoglycine, an
extra electron density was identified in the proximity of the
bound Mn2ϩ ion (Fig. 4A and supplemental Fig. S4B). The den-
sity was assigned as that of (S)-ureidoglycine, given that allan-
toate amidohydrolase produces (S)-ureidoglycine in a ste-
reospecific manner (9) and only the (S)-configuration of
ureidoglycine fits the electron density identified in the binary
AtUGlyAH. A, surface representation of the opening in the C domain of
monomer is shown. In this orientation, the N-terminal protruding region is
locatedonthebackside. Theactivesiteresiduesareindicatedwiththebound
Mn2ϩ ioningreen. B, shownaretheMn2ϩ bindingresidues, withtheMn2ϩ ion
in a black sphere and water molecules indicated by red circles. The Mn2ϩ ion
and water molecule trans to His-237 are overlaid with a 2Fo Ϫ Fc electron
density map contoured at 3.0␴ and 1.0␴, respectively. The axial water mole-
cule across from Gln-275 is present in some monomers, and its density is
relativelydisorderedbutobviousat0.8␴ofthe2Fo ϪFc electrondensitymap.
Stereochemical details of the Mn2ϩ binding site are shown in supplemental
Table S3.
Active Site in Ligand-free Form of AtUGlyAH—In the mono- complex. We also modeled the observed density with ureido-
mer, each N and C domain with a funnel-like ␤-barrel structure glycolate, the product of UGlyAH (Scheme 1); however, only
exhibits two possible openings, one at each end of the long axis the (R)-configuration fits the density, which is inconsistent
of the funnel (Fig. 1B). However, various interactions in mono- with the previous assignment of absolute stereochemistry of
mers and octamers effectively seal off these openings, except for the ureidoglycolate enantiomer produced by UGlyAH (32).
one end in the C domain. Specifically, a smaller opening near ␤3 Therefore, it is likely that low catalytic activity of crystalline
and ␤4 in the N domain is occluded by the C-terminal segment AtUGlyAH relative to that in solution as well as the cryo-
from the adjacent monomer within the layer (Fig. 2A and sup- genic conditions during x-ray data collection stabilizes
plemental Fig. S3A). The entrance to a larger opening in the N chemically labile (S)-ureidoglycine rather than catalyzing
domain is closed off by several layers of hydrophobic residues the formation of (S)-ureidoglycolate.
whose side chains point into the cavity from the central and the
There were no significant changes in conformation between
outer side ␤-strands in a cupin fold (supplemental Fig. S4A). In the ligand-free form and the binary complex, with an r.m.s.d. of
contrast, the inside cavity of the C domain is readily accessible less than 0.5 and 0.32 Å for 258 and 2064 C␣ atoms in the
from the surface of the enzyme through the larger opening, monomer and octamer, respectively. When bound to the Mn2ϩ
with its location in the direction of the dorsal surface in the coordination shell, (S)-ureidoglycine is in an almost planar con-
layer (Fig. 1B). A smaller end of the C domain funnel points formation (Fig. 4, A and B); the carboxyl and the ureido groups
toward the center of the octamer but is sealed off by the pres- are in an anti conformation along the bond between the chiral
ence of ␤1 and ␤2 (Fig. 1B).
carbon atom and the nitrogen atom in the amide bond, with a
Consistent with the requirement of Mn2ϩ ion for catalytic dihedral angle of 162° (Ϯ16.0) throughout the 16 monomers.
activity (9), the location of the active site in AtUGlyAH was The amino group and hydrogen attached to the chiral carbon
indicated by the presence of a metal binding site in the C atom are the only ones out of the plane. The substrate (S)-
domain. Indeed, crystallization of AtUGlyAH was performed ureidoglycine is almost parallel to the axial plane of the Mn2ϩ
with 1 mM MnCl2. The metal binding site is embedded in the coordination shell (Fig. 4B); the ureido group is located at the
middle of a funnel-like cavity in the C domain, which is located innermost part of the active site, whereas the carboxylate group
at 14 Å deep from the surface (Fig. 3A). Four residues from the points toward an entrance to the active site. Specifically, the
central side and the outer side of a ␤-barrel are involved in the binding of (S)-ureidoglycine is achieved by replacing the two
metal coordination shell with one or two water molecules water molecules (one each for the axial and equatorial ligands in
depending on the subunit (Fig. 3B). Specifically, Glu-235 and the Mn2ϩ coordination shell of the ligand-free form). One of
His-237 in the long loop between ␤13 and ␤14, which trans- the oxygen atoms in the carboxylate group and the nitrogen
verses and folds back between the central and outer side of the atom in the amide bond then take the positions of the axial and
␤-barrel, as well as His-241 in the central ␤14 and Gln-275 in equatorial ligands, respectively (Fig. 4B), without distorting the
the outer side ␤18 are part of an octahedral coordination with geometry of the octahedral coordination with the bound metal
the bound metal. In this coordination, the metal ion lies in the (supplemental Table S3B).
center of an equatorial plane formed by Glu-235, His-237, His-
The binding of (S)-ureidoglycine in the complex is further
241, and a water molecule across from His-237 (Fig. 3B and stabilized by hydrogen bonding along the molecule (Fig. 4, B
supplemental Table S3A). Gln-275 and another water molecule and C). Specifically, the carboxyl group of (S)-ureidoglycine
trans to Gln-275 serve as the axial ligands, which are normal to mediates several interactions within 3.0 Å, i.e. a possible charge
the equatorial plane; the electron density for the axial water neutralization with Lys-291 and hydrogen bonding with an
molecule is relatively disordered in different monomers. Under equatorial ligand His-241. The side chain of an equatorial
MAY 25, 2012•VOLUME 287•NUMBER 22
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
FIGURE 4. The binding mode of (S)-ureidoglycine in the binary complex. A, the modeled (S)-ureidoglycine is overlaid with an Fo Ϫ Fc electron density map
contoured at 2.5␴. B, the active site in the binary complex is shown in a stereoscopic view, with neighboring residues less than 5.0 Å from the bound
(S)-ureidoglycine. C, shown is a schematic representation of the interactions around the Mn2ϩ ion and (S)-ureidoglycine. Residues involved in hydrophobic
interactions and hydrogen bonding are indicated in magenta and black, respectively, with the interatomic distance values averaged among 16 monomers. For
clarity, the following possible hydrogen bonds were not indicated: the ring nitrogen of His-241 to the carboxyl group of (S)-ureidoglycine within 3.2 Å, the
side-chain carbonyl oxygen of Gln-275 to the amino group of the ureido group within 2.8 Å, and the two hydroxyl groups in Tyr-252 and Tyr-287 within 3.7 Å.
ligand Glu-235 also stabilizes the binding of the amino group allantoate amidohydrolase (11), with allantoin as the substrate
within a distance of 2.8 Å and the nitrogen atom in the amide (supplemental Figs. S1 and S2B). We also used a coupled reac-
bond within a distance of 3.0 Å, allowing the (S) configuration tion in which (S)-ureidoglycolate produced by AtUGlyAH was
of ureidoglycine. The ureido group is also within a hydrogen- oxidized into oxalurate by an NAD-dependent E. coli (S)-ure-
bonding distance of 2.8–3.0 Å from Tyr-252, Tyr-287, and an idoglycolate dehydrogenase, with concomitant formation of
axial ligand Gln-275, facilitating the binding of the ureido group NADH (supplemental Figs. S1 and S2C). Therefore, the
in the innermost part of the active site. In addition, hydropho- steady-state kinetic parameters for various AtUGlyAH mutant
bic residues comprise a part of the active site within 5.0 Å from enzymes were measured by monitoring the increase in absor-
the bound substrate and position the incoming (S)-ureidogly- bance at 340 nm caused by the formation of NADH.
cine into the productive binding mode for catalysis. These res-
A total of eight residues were selected for site-directed
idues include Phe-204, Met-223, and Leu-231 near the ureido mutagenesis (Fig. 4C); their kinetic parameters are listed in
group and Met-269 and Leu-289 near the amide bond (Fig. 4, B Table 1 (supplemental Fig. S5). The residues involved in the
and C). It is noteworthy that the Mn2ϩ binding residues and Mn2ϩ coordination shell were sensitive to mutation. Mutant
those hydrophilic residues near the (S)-ureidoglycine binding site enzymes such as E235A, H237A, H241A, and Q275A were cat-
are invariant among UGlyAHs and its structural homologs (Fig. alytically incompetent, suggesting that these residues are essen-
1A), suggesting the functional roles of these residues in catalysis. In tial for coordinating with the Mn2ϩ ion. It is noticeable that
particular, His-221, which is conserved in AtUGlyAH and its E235Q mutant is an essentially inactive enzyme (Table 1) but is
E. coli ortholog (Fig. 1A; PDB ID 1RC6), is located on the side of capable of binding the Mn2ϩ ion as much as does the WT
the hydrogen attached to the chiral carbon atom, 5.5 Å from the enzyme (supplemental Table S4). We did not expect to find that
chiral carbon atom.
the Km value of Y252F was comparable, but its kcat value was
Functional Analysis—We produced (S)-ureidoglycine in situ reduced by ϳ10-fold compared with that of WT enzyme. How-
by employing the two preceding enzymes, allantoinase (14) and ever, enzyme activity was essentially abolished in the Y287F and
18802 JOURNAL OF BIOLOGICAL CHEMISTRY
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(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
TABLE 1
ond, the resulting binding mode serves as the molecular basis of
the catalytic enantioselectivity. More specifically, a molecular
face with a hydrogen atom attached to the chiral carbon atom is
vulnerable to enzyme catalysis. Of the neighboring residues,
Tyr-287 and Lys-291 are essential for enzyme activity, possibly
by dictating the orientation of the ureido and carboxyl groups
of the substrate, respectively (Table 1 and Fig. 4B). Binding of
the (S) configuration of ureidoglycine is further stabilized by
Glu-235 due to possible interactions between the amino group
of a reaction center carbon and the carboxyl group of Glu-235
(Fig. 4C). In addition, the side chain carboxyl group on Glu-235
appears to be crucial in catalysis because the E235Q mutant still
containing Mn2ϩ ion (supplemental Table S4) is catalytically
incompetent.
Kinetic parameters of AtUGlyAH and its mutants
Mutants as follows were inactive: E235A, E235Q, H237A, H241A, Q275A, Y287A,
Y287F, K291A. The initial velocity measurements of the H221A mutant were
unsuccessful, mainly due to non-linear changes in absorbance as a function of reac-
tion time under various assay conditions.
Km
mM
kcat
s
761 (14.1)
73 (0.73)
493 (10.9)
k
cat/Km
Ϫ1
Ϫ1
mMϪ1s
429
32
WT
Y252F
K291R
1.77 (0.104)a
2.30 (0.0910)
3.46 (0.196)
143
a Values in parentheses are S.E. estimated by the program SigmaPlot.
Y287A mutants, suggesting that the side chain hydroxyl group
in Tyr-287, but not Tyr-252, plays an essential role in catalysis
and/or substrate binding. The Km value of K291R was ϳ2-fold
higher, and its kcat value was ϳ65% that of the WT enzyme,
whereas enzyme activity was completely absent in the K291A
mutant. This suggests that the positive charge at Lys-291 plays
a pivotal role in substrate binding, possibly by neutralizing the
negative charge of the carboxyl group of (S)-ureidoglycine (Fig.
4, B and C). Unlike other mutant enzymes, initial velocity mea-
surements of the H221A mutant were unsuccessful, mainly due
to nonlinear changes in absorbance as a function of reaction
time under various assay conditions. However, its activity was
estimated to be similar to that of Y252F (supplemental Fig.
S5D). Because all AtUGlyAH mutant enzymes used in this
functional analysis were estimated to be in a native conforma-
tion based on circular dichroism spectra that was similar to the
WT enzyme (supplemental Fig. S6), a lack of activity in some of
these mutants is related to the functional features of those res-
idues, not due to a possible conformation change by mutation.
We considered potential catalytic mechanisms of
AtUGlyAH by examining structural and functional analyses as
well as inversion of configuration at the chiral carbon center
between (S)-ureidoglycine and (S)-ureidoglycolate (Scheme 1)
(9). A possible direct attack of the general base-catalyzed water
molecule at the chiral carbon appears to be highly unlikely; such
an Sn2-type reaction requires a water molecule that presents on
the side opposite to the leaving amino group, with a geometrical
requirement for an inline attack on the chiral carbon center.
The presence of the water molecule is implausible, mainly due
to steric hindrance in that area which is densely populated with
His-241, Leu-289, and Lys-291 (Fig. 4B). Alternatively, allantu-
rate (5) could be an intermediate in catalysis (supplemental Fig.
S7). Glu-235, with the probable low pK value of the hydrogen on
the Mn-bound amide nitrogen, could facilitate abstraction of
the proton from the amide nitrogen and subsequent transfer of
this proton to the amino leaving group in the form of ammonia,
which is analogous to the proposed function of the metal-
DISCUSSION
The presence of a gene encoding the UGlyAH enzyme and its bound aspartate in adenine deaminase (34). Subsequently, the
biochemical function in the ureide pathway were only recently resulting planar intermediate allanturate could be subject to a
identified in both A. thaliana and E. coli (9, 10). However, the nucleophilic attack by the general base-catalyzed water mole-
Research Centers for Structural Genomics had earlier deter- cule, yielding (S)-ureidoglycolate. Specifically, it is likely that
mined the crystal structure of YlbA, an E. coli ortholog of the re face of the reaction center carbon in the intermediate
UGlyAH (Fig. 1A; PDB ID 1RC6), and three structural remains occluded by Glu-235, as observed in the binary com-
homologs from other bacteria (Fig. 1A; PDB IDs 1SFN, 1SEF, plex (Fig. 4B). Therefore, the general base-catalyzed water mol-
and 1SQ4). These four proteins exhibit structural features ecule could reach the intermediate only from the si face of the
highly homologous with those of AtUGlyAH; a bicupin mono- planar intermediate, which is consistent with a previous obser-
mer and an overall octameric conformation, with Z-scores of vation of an inversion of configuration at the chiral carbon
28.3–34.8, sequence identities of 26–40%, and r.m.s.d. values of atom after catalysis (Scheme 1) (9).
1.4–2.5 Å for the monomer, as defined by the DALI program
However, this analysis does not reveal any obvious general
(33). Although the metal binding site was not recognized in base to deprotonate the incoming water molecule in the imme-
those structures, high similarities in both structure and diate vicinity of the chiral carbon atom of (S)-ureidoglycine
sequence, including residues for metal coordination and the (Fig. 4, B and C) but provides two candidates, His-221 and Tyr-
substrate binding site (Fig. 1A), suggest that these three struc- 287. Both are located on the side opposite the leaving amino
tural homologs are also members of the UGlyAH family. In group of the substrate, 5.3–5.4 Å from the chiral carbon atom.
particular, the cupin fold in this family of proteins is associated The presence of a water molecule connecting His-221 and the
with a broad range of enzyme activities and is widely distributed chiral carbon atom (Fig. 4C) as well as the low catalytic activity
in the protein kingdom (31).
of the H221A mutant (supplemental Fig. S5D) raises the possi-
Structural and functional analysis of AtUGlyAH revealed bility that His-221 acts as the proposed general base. However,
several functional consequences. First, AtUGlyAH is a Mn2ϩ
-
His-221 is not a strictly conserved residue in the UGlyAH fam-
dependent enzyme (9). It is evident from the structure of the ily (Fig. 1A), in which the mechanistic features should remain
binary complex that the Mn2ϩ ion acts as a molecular anchor identical. In contrast, Tyr-287 is invariant in the family, but its
for the substrate; it positions the incoming (S)-ureidoglycine side chain hydroxyl group should be deprotonated to act as a
into the productive binding mode for catalysis (Fig. 4B). Sec- general base. The following observations suggest that a hydro-
MAY 25, 2012•VOLUME 287•NUMBER 22
JOURNAL OF BIOLOGICAL CHEMISTRY 18803

(S)-Ureidoglycine Aminohydrolase in Purine Catabolism
10. Werner, A. K., Romeis, T., and Witte, C. P. (2010) Ureide catabolism in
gen bonding network between Tyr-252 and Tyr-287 as well as
the ureido group might participate in the deprotonation of Tyr-
287 and facilitate its functional role in catalysis; (i) the Y287F
mutant is an inactive enzyme, (ii) the two hydroxyl groups in
Tyr-287 and Tyr-252 are separated by ϳ3.7 Å, and (iii) the
hydroxyl group in Tyr-252 is involved in catalysis rather than
substrate binding, given that the Y252F mutation largely affects
the kcat, but not the Km, value. Therefore, Tyr-287 is a candidate
general base. However, Tyr-287 became almost buried after
binding of (S)-ureidoglycine. Lack of the amino group in the
intermediate could cause its binding mode to differ from that in
the enzyme-substrate complex, possibly by inclining toward
Glu-235, exposing Tyr-287 to the solvent. Further investigation
is necessary for conclusive identification of the general base in
AtUGlyAH.
Arabidopsis thaliana and Escherichia coli. Nat. Chem. Biol. 6, 19–21
11. Agarwal, R., Burley, S. K., and Swaminathan, S. (2007) Structural analysis
of a ternary complex of allantoate amidohydrolase from Escherichia coli
reveals its mechanics. J. Mol. Biol. 368, 450–463
12. Cendron, L., Berni, R., Folli, C., Ramazzina, I., Percudani, R., and Zanotti,
G. (2007) The structure of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazo-
line decarboxylase provides insights into the mechanism of uric acid deg-
radation. J. Biol. Chem. 282, 18182–18189
13. Kim, K., Park, J., and Rhee S. (2007) Structural and functional basis for
(S)-allantoin formation in the ureide pathway. J. Biol. Chem. 282,
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14. Kim, K., Kim, M. I., Chung, J., Ahn, J. H., and Rhee, S. (2009) Crystal
structure of metal-dependent allantoinase from Escherichia coli. J. Mol.
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15. Ramazzina, I., Cendron, L., Folli, C., Berni, R., Monteverdi, D., Zanotti, G.,
and Percudani, R. (2008) Logical identification of an allantoinase analog
(puuE) recruited from polysaccharide deacetylases. J. Biol. Chem. 283,
23295–23304
In this study we determined a crystal structure of the ligand-
free form of AtUGlyAH and its complex with (S)-ureidoglycine.
The structures revealed the overall features of both the mono-
meric and octameric conformations, which are applicable to
other UGlyAH family proteins, as well as the binding mode of
(S)-ureidoglycine in the Mn2ϩ coordination shell. Our enzyme
assay, which employed an E. coli (S)-ureidoglycolate dehydro-
genase as a coupled enzyme, allows for measurement of the
steady-state kinetic parameters of AtUGlyAH. These results
provide molecular insights into the structure of UGlyAH and
early events in the catalytic release of ammonia from the nitro-
gen-rich metabolite, which is the crucial step of the ureide
pathway.
16. Hennebry, S. C. (2009) Evolutionary changes to transthyretin. Structure
and function of a transthyretin-like ancestral protein. FEBS J. 276,
5367–5379
17. French, J. B., and Ealick, S. E. (2010) Structural and mechanistic studies on
Klebsiella pneumoniae 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
decarboxylase. J. Biol. Chem. 285, 35446–35454
18. Reumann, S., Babujee, L., Ma, C., Wienkoop, S., Siemsen, T., Antonicelli,
G. E., Rasche, N., Lu¨der, F., Weckwerth, W., and Jahn, O. (2007) Proteome
analysis of Arabidopsis leaf peroxisomes reveals novel targeting pep-
tides, metabolic pathways, and defense mechanisms. Plant Cell 19,
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19. Lamberto, I., Percudani, R., Gatti, R., Folli, C., and Petrucco, S. (2010)
Conserved alternative splicing of Arabidopsis transthyretin-like deter-
mines protein localization and S-allantoin synthesis in peroxisomes. Plant
Cell 22, 1564–1574
20. Otwinowski, Z., and Minor, W. (1997) Processing of x-ray diffraction data
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21. Terwilliger, T. C., and Berendzen, J. (1999) Automated MAD and MIR
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Acknowledgments—We thank Woo-Suk Jung for his contribution to
the initial stage of research. We also thank Peter Tipton for his sug-
gestion for the proposed mechanism and Hanjin Oh for performing the
circular dichroism measurements.
23. Terwilliger, T. C. (2003) Automated main-chain model building by tem-
plate matching and iterative fragment extension. Acta Crystallogr. D Biol.
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MAY 25, 2012•VOLUME 287•NUMBER 22
JOURNAL OF BIOLOGICAL CHEMISTRY 18805

Protein Structure and Folding:
Structural and Functional Insights into (S
)-Ureidoglycine Aminohydrolase, Key
Enzyme of Purine Catabolism in
Arabidopsis thaliana
Inchul Shin, Riccardo Percudani and Sangkee
Rhee
J. Biol. Chem. 2012, 287:18796-18805.
doi: 10.1074/jbc.M111.331819 originally published online April 5, 2012
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