Characterization of the structure and function of Klebsiella pneumoniae allantoin racemase

Article abstract of DOI:10.1016/j.jmb.2011.05.016

The oxidative catabolism of uric acid produces 5-hydroxyisourate (HIU), which is further degraded to (S)-allantoin by two enzymes, HIU hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase. The intermediates of the latter two reactions, HIU and 2-oxo-4-hydroxy-4-carboxy-5- ureidoimidazoline, are unstable in solution and decay nonstereospecifically to allantoin. In addition, nonenzymatic racemization of allantoin has been shown to occur at physiological pH. Since the further breakdown of allantoin is catalyzed by allantoinase, an enzyme that is specific for (S)-allantoin, an allantoin racemase is necessary for complete and efficient catabolism of uric acid. In this work, we characterize the structure and activity of allantoin racemase from Klebsiella pneumoniae (KpHpxA). In addition to an unliganded structure solved using selenomethionyl single-wavelength anomalous dispersion, structures of C79S/C184S KpHpxA in complex with allantoin and with 5-acetylhydantoin are presented. These structures reveal several important features of the active site including an oxyanion hole and a polar binding pocket that interacts with the ureido tail of allantoin and serves to control the orientation of the hydantoin ring. The ability of KpHpxA to interconvert the (R)- and (S)-enantiomers of allantoin is demonstrated, and analysis of the steady-state kinetics of KpHpxA yielded a k_cat/K_m of 6.0 × 10⁵ M^{- 1} s^{- 1}. Mutation of either of the active-site cysteines, Cys79 or Cys184, to serine inactivates this enzyme. The data presented provide new insights into the activity and substrate specificity of this enzyme and enable us to propose a mechanism for catalysis that is consistent with the two-base mechanism observed in other members of the aspartate/glutamate family.

Full text of DOI:10.1016/j.jmb.2011.05.016

doi:10.1016/j.jmb.2011.05.016
J. Mol. Biol. (2011) 410, 447–460
Contents lists available at www.sciencedirect.com
Journal of Molecular Biology
journal homepage: http://ees.elsevier.com.jmb
Characterization of the Structure and Function of
Klebsiella pneumoniae Allantoin Racemase
Jarrod B. French1, David B. Neau1,2 and Steven E. Ealick1
⁎
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
2NE-CAT, Argonne National Laboratory, Argonne, IL 60439, USA
Received 29 March 2011;
received in revised form
4 May 2011;
accepted 10 May 2011
Available online
17 May 2011
The oxidative catabolism of uric acid produces 5-hydroxyisourate (HIU),
which is further degraded to (S)-allantoin by two enzymes, HIU hydrolase
and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase. The
intermediates of the latter two reactions, HIU and 2-oxo-4-hydroxy-4-
carboxy-5-ureidoimidazoline, are unstable in solution and decay nonster-
eospecifically to allantoin. In addition, nonenzymatic racemization of
allantoin has been shown to occur at physiological pH. Since the further
breakdown of allantoin is catalyzed by allantoinase, an enzyme that is
specific for (S)-allantoin, an allantoin racemase is necessary for complete
and efficient catabolism of uric acid. In this work, we characterize the
structure and activity of allantoin racemase from Klebsiella pneumoniae
(KpHpxA). In addition to an unliganded structure solved using seleno-
methionyl single-wavelength anomalous dispersion, structures of C79S/
C184S KpHpxA in complex with allantoin and with 5-acetylhydantoin are
presented. These structures reveal several important features of the active
site including an oxyanion hole and a polar binding pocket that interacts
with the ureido tail of allantoin and serves to control the orientation of the
hydantoin ring. The ability of KpHpxA to interconvert the (R)- and (S)-
enantiomers of allantoin is demonstrated, and analysis of the steady-state
kinetics of KpHpxA yielded a kcat/Km of 6.0×105 M−1 s−1. Mutation of
either of the active-site cysteines, Cys79 or Cys184, to serine inactivates this
enzyme. The data presented provide new insights into the activity and
substrate specificity of this enzyme and enable us to propose a mechanism
for catalysis that is consistent with the two-base mechanism observed in
other members of the aspartate/glutamate family.
Edited by G. Schulz
Keywords:
purine catabolism;
ureide;
X-ray crystallography;
racemization;
hydantoin
© 2011 Elsevier Ltd. All rights reserved.
some bacteria to provide a source of nitrogen,
particularly when other nitrogen sources are depleted
(Scheme 1).3,4 The first stage of this pathway involves
Introduction
The enzymatic degradation of uric acid is the final
stage of purine catabolism and serves in plants and
three enzymes and facilitates the stereospecific
oxidative decomposition of uric acid to (S)-allanto-
in. Upon oxidation by urate oxidase, two additional
enzymes, 5-hydroxyisourate (HIU) hydrolase and 2-
oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
(OHCU) decarboxylase, complete the conversion
from urate to (S)-allantoin.5–7 While the product of
these enzymatic conversions is solely the (S)-
enantiomer, nonenzymatic decomposition of both
HIU and OHCU can occur and does so
*Corresponding author. 120 Baker Lab, Cornell
University, Ithaca, NY 14853-1301, USA. E-mail address:
see3@cornell.edu.
Abbreviations used: HIU, 5-hydroxyisourate; OHCU, 2-
oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline; SeMet,
selenomethionyl; NE-CAT, Northeastern Collaborative
Access Team; PDB, Protein Data Bank.
0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

448
Klebsiella pneumoniae Allantoin Racemase
Scheme 1. Purine catabolic pathway in K. pneumoniae.1,2
nonstereospecifically. In addition, slow nonenzy-
matic racemization of allantoin has been observed
to occur.8
residues.18,19 Binding and kinetic studies combined
with site-directed mutagenesis have demonstrated
the importance of these two cysteines for both
substrate recognition and catalysis. While this
enzyme can bind and catalyze the conversion of
both isomers, it has been reported that it does so
with differing specificity. Much of the binding and
catalytic selectivity for the different isomers have
been attributed to the two active-site cysteine
residues.19,20
In this work, we present the first X-ray crystal
structures of allantoin racemase in both unli-
ganded and ligand-bound forms. These structures
from the opportunistic human pathogen Klebsiella
pneumoniae provide valuable insights into substrate
binding and selectivity. In addition, the KpHpxA-
catalyzed conversion of either (R)- or (S)-allantoin
to the racemic mixture is demonstrated using
circular dichroism (CD) spectroscopy, and the
steady-state kinetic parameters are reported. Mu-
tation of either of the active-site cysteine residues
to serine completely abolishes the ability of the
enzyme to catalyze the racemization of allantoin.
Examination of the structural and biochemical data
provided herein suggests that this enzyme utilizes
a conserved two-base mechanism to catalyze the
racemization of allantoin.
The conversion of allantoin to allantoate by an
allantoinase enzyme is a point of convergence in
the metabolism of ureides.3,4,9 This step is present
in all organisms that catabolize allantoin. To date,
two types of allantoinase have been identified, a
metal-dependent form and a metal-independent
form.10–12 While these two enzymes may differ in
mechanism, they both preferentially bind the (S)-
enantiomer of allantoin. Considering the selectivity
of the allantoinase enzymes and the various
mechanisms operating to produce racemic mix-
tures of allantoin, efficient use of this molecule
would require the presence of a racemase enzyme.
Recent genetic studies on Klebsiella sp. have
identified a gene cluster for purine utilization
that encodes several enzymes that catalyze novel
chemical transformations, including an enzyme
that was putatively identified as an allantoin
racemase.1,2 This enzyme shares 45% identity
over 96% of its length with the hydantoin
racemase from Pseudomonas sp.
While enzymatic racemization of allantoin was
noted as early as the 1970s,13–15 there has been very
little reported in the subsequent three decades.
Despite this paucity of data on allantoin racemase,
some progress has been made on the characteriza-
tion of the homologous hydantoin racemase.16 This
enzyme belongs to the aspartate/glutamate race-
mase superfamily and is known to catalyze
racemization of 5-substituted hydantoins in a
cofactor- and metal-independent fashion. Hydan-
toin racemase, which catalyzes the racemization of
various 5-substituted hydantoins, is a commercially
important biocatalyst used in the production of
optically pure amino acids.17
Results
Structure of unliganded KpHpxA
The structure of KpHpxA was determined to 2.1 Å
resolution using selenomethionyl (SeMet) single-
wavelength anomalous dispersion phasing. Other
than the polyhistidine tag and the two N-terminal
residues, the entire protein was modeled.
KpHpxA is an α/β protein with two domains
that form a V-shape in the protomer (Fig. 1a). The
two domains share a high degree of similarity
Hydantoin racemase and other members of the
aspartate/glutamate racemase superfamily are be-
lieved to operate using a two-base mechanism that
involves a pair of highly conserved cysteine

Klebsiella pneumoniae Allantoin Racemase
449
Fig. 1. Structure of KpHpxA. (a) KpHpxA protomer. (b) Hexameric structure of KpHpxA seen in the crystals and
confirmed by size-exclusion chromatography. (c) Topology diagram of KpHpxA. Helixes are shown as blue cylinders,
and strands are shown as green arrows. The numbers correspond to the residue numbers of the first and last amino acids
of each secondary structure element. The N and C termini are marked with an N and a C, respectively.
(RMSD, 3.1 Å), and each contains a four-stranded
parallel β-sheet sandwiched between two pairs of
α-helices (Fig. 1a and c). When crystallographic
symmetry-related molecules were considered, the
crystallographic data suggested that KpHpxA
formed a stable hexamer in the crystal (Fig. 1b).
The quaternary structure was verified by size-
exclusion chromatography (data not shown). The
hexamer is made up of a trimer of dimers, with the
three identical dimer interfaces occurring at the
groove between domains. This interface has an
approximate buried surface area of 3400 Å2 and is
dominated by polar contacts between amphipathic
helices 4 and 9 of the two protomers. The interface
Fig. 2. Active site of KpHpxA. (a) Stereo diagram of the active site of KpHpxA in complex with allantoin. The structure
shown (carbon atoms in green, nitrogen atoms in blue and oxygen atoms in red) is that of the C79S/C184S double mutant
with allantoin bound (C in black, N in blue and O in red). Waters are represented as red spheres and are labeled with “w.”
Putative hydrogen bonds are illustrated with broken lines. (b) Schematic drawing of the active-site contacts between
KpHpxA and allantoin. Broken lines indicate putative hydrogen bonds. Cys79 and Cys184 are not shown.

450
Klebsiella pneumoniae Allantoin Racemase
between the dimers is predominantly along helices
2 and 3 and has only slightly less buried surface
area (approximately 3340 Å2) than the dimer
interface itself. In addition, the 20 C-terminal
residues form a loop that packs along the length
of helix 2 of the neighboring molecule, providing
additional polar contacts. Overall, this leads to a
tightly packed hexamer with a total buried surface
area of approximately 22,000 Å2.
The active site of KpHpxA, positively identified
by the presence of allantoin or 5-acetylhydantoin in
KpHpxA–ligand complexes (see below), is occu-
pied predominantly by water molecules in the
absence of ligand. In addition, a spherical region of
strong electron density was observed between the
backbone amides of Phe80 and Gly185, putatively
identified as the oxyanion hole (see below). This
density was modeled as a chloride ion in the
unliganded structure of KpHpxA.
density best fit the enol form of allantoin (Fig. 2b).
Most of the interactions between the enzyme and the
ligand are made through the backbone of KpHpxA.
A notable exception to this is a region surrounding
the amide tail of the ligand where several hydrogen
bonds are made to the side chains of Thr118 and
Thr119, the backbone of Gly183 and a water
molecule positioned by Thr123.
The hydantoin ring of allantoin is positioned in the
active site through three sets of interactions. In
addition to the aforementioned binding pocket that
tightly holds the allantoin tail in place, additional
interactions are made through the two oxygen atoms
of the hydantoin ring. These are each held in place by
a pair of residues: the backbone amide nitrogens of
Phe80 and Gly185 for the C4 oxygen atom and the
backbone amide nitrogen of Ile47 and the side chain
of Asn12 for the C2 oxygen atom (Fig. 2b). Both of
these pairs of hydrogen-bonding interactions could
also provide the necessary stabilizing interactions
required for an oxyanion hole. Considering the
geometry of the ligand in the active site, however,
it is likely that only the former pair of residues would
function in this capacity during catalysis.
C79S/C184S KpHpxA–allantoin complex structure
In order to examine active-site interactions in
KpHpxA, we constructed the C79S/C184S double
mutant and determined the structure in complex
with allantoin. The observed difference density in
the active site allowed for the unambiguous
placement of the ligand (Figs. 2a and 3a). While
the solution used for soaking the crystals was a
mixture of the (R)- and (S)-isomers, the observed
The two conserved cysteine residues, Cys79 and
Cys184, which are replaced by serine residues in the
mutant structure, are situated on opposite sides of
allantoin in the asymmetric active site of KpHpxA
(Fig. 3a). In the structure of the C79S/C184S
KpHpxA double mutant with allantoin bound, the
Fig. 3. Ligands bound to KpHpxA. (a) Stereo diagram of allantoin bound to the C79S/C184S KpHpxA double mutant.
(b) Stereo diagram of 5-acetylhydantoin bound to the C79S/C184S KpHpxA double mutant. In both cases, carbon atoms
are in green, oxygen atoms are in red and nitrogen atoms are in blue. The red sphere labeled with “w” is a water molecule.
The side chain of Phe80 has been omitted for clarity. The electron density in both cases is from difference maps generated
before the addition of the ligand molecule into the model and is contoured at 3 σ.

Klebsiella pneumoniae Allantoin Racemase
451
Fig. 4. Superposition of KpHpxA and the hydantoin racemase from P. horikoshii (2EQ5). (a) Secondary structural
superposition of the protomers from KpHpxA (white) and 2EQ5 (green). (b) Stereo diagram of the superposition of the
active sites of KpHpxA (white carbon atoms, blue nitrogen atoms and red oxygen atoms) and 2EQ5 (green carbon atoms)
with allantoin shown (yellow carbon atoms). The two active-site cysteines (serines in the KpHpxA–allantoin structure) are
labeled with C/S, the putative oxyanion hole is designated with an O and each of the threonine residues that interact with
the “tail” region of allantoin in KpHpxA is labeled with T.
electron density for the Ser79 side chain was diffuse
and was best fit by two rotamers, each having 50%
occupancy (Fig. 3a). The distances between the two
serine residues and C5 of allantoin in the structure
are 3.2 Å for Ser79 (rotamer closest to allantoin) and
4.2 Å for Ser184. Also present in the active site is a
water molecule located near Ser79 (Fig. 3a). This
water molecule is 2.6 Å from Ser79 and 4.3 Å from
C5 of allantoin. The water molecule also makes
hydrogen bonds with the backbone nitrogen of
Gly81 and the side chain of Glu53.
the conserved cysteine residues and in the putative
oxyanion hole. Beyond these features, little similar-
ity exists. Not surprisingly, the tail-binding region of
the KpHpxA active site, which is populated by
several threonine residues, is not present in the
hydantoin racemase structure. In 2EQ5, this region
is composed of predominantly nonpolar residues,
presumably to accommodate the typically aliphatic
chains present in the 5-substituted hydantoin sub-
strates. Additional interactions observed between
allantoin and KpHpxA, particularly at the C2
oxygen atom, are not present in 2EQ5. Overall, the
active site of the hydantoin racemase is larger and
more open than that observed in the allantoin
racemase structure of KpHpxA.
Structural similarity to other proteins
A search for structurally homologous proteins
using the Dali21 server yielded several related
structures (Supplementary Table S1). As expected,
all of these structures belonged to the aspartate/
glutamate racemase superfamily of proteins. The
protein of known structure that is most similar to
KpHpxA is the hydantoin racemase from Pyro-
coccus horikoshii [Protein Data Bank (PDB) identi-
fier 2EQ5; unpublished]. The asymmetric unit
contains a dimer, and higher oligomerization is
not allowed in the hexagonal space group P65.
The orientation of the two protomers in the
dimeric 2EQ5 structure is similar to that of a
dimer from the KpHpxA hexamer. Primarily
smaller, hydrophobic residues populate the 2EQ5
interface, resulting in a slightly smaller separation
between the protomers compared to KpHpxA. A
structural superposition of KpHpxA and 2EQ5
shows that the two proteins contain very similar
secondary structure elements, although some of
the helical regions do not align well (Fig. 4a). In
spite of the overall similarity of the two proteins,
the active sites display some distinct differences
(Fig. 4b).
C79S/C184S KpHpxA–5-acetylhydantoin
complex structure
To further probe the active site of KpHpxA and
examine how the residues contained within it
determine specificity, we determined the structure
of KpHpxA in complex with the allantoin
analogue 5-acetylhydantoin. Unlike allantoin,
which is observed as an enol in the active site,
the (R)-enantiomer [equivalent to the (S)-enantio-
mer of allantoin] is clearly bound to KpHpxA in
this structure (Fig. 3b). While the orientation of
the hydantoin ring is similar for the two ligands,
the tail region makes distinctly different binding
interactions. The substitution of a carbon for N6
of allantoin leads to a different geometry and
moves the acetyl group away from the threonine-
rich pocket of the active site. This causes a slight
shift in the ring and leads to additional hydrogen
bonding between the acetyl group of 5-acetylhy-
dantoin and Ser184 of the enzyme. A similar
feature in both the KpHpxA–allantoin and
KpHpxA–5-acetylhydantoin structures is the elec-
tron density for the Ser79 side chain, which is
Superposition of the active-site residues of
KpHpxA and 2EQ5 shows expected similarities in

452
Klebsiella pneumoniae Allantoin Racemase
consistent with the presence of two rotamers, each
having approximately 50% occupancy.
allowed for the facile measurement of this reaction.
As shown in Fig. 5, KpHpxA catalyzes the racemiza-
tion of allantoin. The rates of racemization of (S)- and
(R)-isomers occur at 183 17 s−1 and 238 19 s−1
,
Racemization of (R)- and (S)-allantoin by
KpHpxA
respectively, 6 orders of magnitude faster than the
nonenzymatic racemization reaction (2×10−4 s−1
;
Supplementary Fig. S1). A fit of initial rates of reaction
to the Michaelis–Menten equation yielded Km values
of 0.3 0.1 mM and 0.4 0.1 mM for the (S)- and (R)-
isomers, respectively. The average kcat/Km for the
To demonstrate that KpHpxA functions as an
allantoin racemase, we tested the ability of this
enzyme to catalyze the conversion of each of the
isolated enantiomers to the racemic mixture. The
difference in CD spectra of the two enantiomers
KpHpxA-catalyzed reaction is 6.0×105 M−1 s−1
.
The C79S, C184S and double mutant C79S/C184S
KpHpxA were also tested for their ability to catalyze
the racemization of allantoin. None of the mutant
enzymes tested increased the rate of racemization
over the nonenzymatic rate for either (S)- or (R)-
allantoin (data not shown).
Docking of (R)- and (S)-isomers of allantoin into
the KpHpxA active site
To better understand the possible substrate in-
teractions with KpHpxA, we modeled both (R)- and
(S)-allantoin into the active site of this enzyme. After
the positioning of each isomer by superposition with
the enol form of allantoin observed in the C79S/
C184S KpHpxA structure, the ligand was energy
minimized while fixing the protein side chains. An
overlay of the results with the structure of the
double mutant KpHpxA structure with allantoin
bound is shown in Fig. 6.
Discussion
Overall structure of KpHpxA
KpHpxA is a two-domain, α/β protein that is
structurally homologous to members of the Glu/
Asp racemase superfamily. The two domains of this
enzyme share the same secondary structural ele-
ments, suggesting that a gene duplication and
fusion event occurred at some point in this protein's
evolutionary history (Fig. 1a and c). A clear
hexameric quaternary structure was observed in
the crystal structures of this enzyme and was
confirmed by size-exclusion chromatography (data
not shown). The most structurally similar enzyme,
hydantoin racemase from the archaeon P. horikoshii
(PDB ID 2EQ5), was observed as a dimer in the
crystal structure. While all of the necessary active-
site contacts to the ligands are made by a single
protomer in KpHpxA, a very tight dimer, similar to
what is observed in the hydantoin racemase
structure, is formed between neighboring protomers
in the hexameric molecule (Fig. 1b). Size-exclusion
chromatography on hydantoin racemases from
other organisms is consistent with either a
Fig. 5. Allantoin racemization catalyzed by KpHpxA.
(a) CD spectra of the racemization of (S)-allantoin (green
curves) and (R)-allantoin (blue curves) to the racemic
mixture (black dotted line) over time. In both cases, the
starting concentration of allantoin was 2 mM, and the
curves represent 20-s time intervals from 0 to 80 s. (b) Time
course of KpHpxA-catalyzed racemization of (S)-allantoin
(open squares, monitored at 208 nm) and (R)-allantoin
(open triangles, monitored at 213 nm) to the racemic
mixture.

Klebsiella pneumoniae Allantoin Racemase
453
Fig. 6. Allantoin isomers in KpHpxA active site. Stereo diagram of the (R)-isomer (white carbon atoms) and the (S)-
isomer (yellow carbon atoms) of allantoin docked in the active site of KpHpxA. The observed distances between the two
active-site cysteines and C5 of the respective modeled allantoin isomers are shown in angstroms. The observed enol form
of allantoin is shown in gray.
hexamer22 or a tetramer.23,24 It is possible that the
hexamer observed in the K. pneumoniae structure
provides some evolutionary advantage in stability
or activity over the dimeric form observed in the
archaeal hydantoin racemase, although further
characterization of the archaeal enzyme would be
required to complete this analysis.
changes upon ligand binding (RMSD of α-carbons
for superposition is 0.2 Å). Other than a slight
difference in the side-chain conformation of Glu53
and the fitting of two rotamers to the density for
Ser79 in the C79S/C184S KpHpxA–allantoin struc-
ture, all of the residues of the liganded structure align
well with those of the unliganded structure. Alter-
nate conformations of Ser79/Cys79 are likely im-
portant for catalysis, while the observed flexibility of
the Glu53 side chain suggests that it may act as a gate
for the relatively inaccessible active site (Fig. 8). A
water molecule is also within hydrogen-bonding
distance from Ser79 and Glu53 in the active site of the
C79S/C184S KpHpxA–allantoin structure. This
water molecule is not likely to directly participate
in catalysis but, instead, may serve to help drive the
movement of Glu53 upon product release.
The observed electron density in the active site for
the C79S/C184S KpHpxA–allantoin structure clear-
ly indicates that allantoin is present in its enol form
(Fig. 3a). This is not a surprising result, considering
that allantoin is known to undergo slow racemiza-
tion in solution via keto–enol tautomerization8 and
that the putative reaction intermediate for the
racemization of allantoin is the enolate. This form
of the ligand is not without precedent and has been
observed in the related structures of K. pneumoniae
OHCU decarboxylase29 and Alcaligenes bronchisepti-
cus arylmalonate decarboxylase.27
Comparison of KpHpxA to other structures
A Dali search for structural homologues of
KpHpxA yielded several proteins of the Glu/Asp
racemase superfamily. The proteins of this family
also share the two-domain, α/β fold that is
observed in KpHpxA. While these proteins have
some degree of structural similarity, there is little
sequence conservation among the members of this
group (Fig. 7). Highly conserved residues include
the two cysteine residues implicated in catalysis
(Fig. 7, blue triangles) and several other residues
that serve predominantly structural roles. These
include two isoleucine residues (Ile6 and Ile9)
occupying the middle of a β-sheet (on β1 strand)
and a conserved proline residue (Pro95) that
facilitates a turn from α3 to β4. Aside from the
catalytic cysteine residues, however, none of the
completely conserved residues appear in the active
site of KpHpxA. It should be noted that the cysteine
residue equivalent to Cys79 in KpHpxA is not
conserved in PDB ID 3EIS, an arylmalonate decar-
boxylase, as these enzymes are reported to proceed
via a mechanism different from that of other
members of the superfamily.
The two active-site cysteine residues, which are
substituted by serine residues in the KpHpxA
double mutant, have been reported to play major
roles in a two-base mechanism proposed for
members of the aspartate/glutamate racemase
superfamily.19,30 In the C79S/C184S KpHpxA–
allantoin structure, these residues are situated on
either side of the ligand (Figs. 2a and 3a). The
distance between the oxygen atoms of Ser79 and
Ser184 in this structure is 6.6 Å (using the rotamer of
KpHpxA active site and ligand binding
A comparison of unliganded and the allantoin-
bound complex of KpHpxA illustrates that the
enzyme does not undergo any major structural

454
Klebsiella pneumoniae Allantoin Racemase
Fig. 7. Sequence alignment of several aspartate/glutamate racemase superfamily members. The sequences were
aligned using Clustal W,25 and the figure was produced using the ESPript server.26 The four sequences are the K.
pneumoniae allantoin racemase (HpxA), P. horikoshii hydantoin racemase (2EQ5), Bordetella bronchiseptica arylmalonate
decarboxylase (3EIS27) and Bacillus anthracis glutamate racemase (2GZM28). Red boxes with white lettering are completely
conserved residues, while boxes with red letters on a white background are regions of sequence similarity. The blue
triangles indicate the two conserved cysteines, and the numbering and secondary structural elements shown correspond
to the KpHpxA structure presented herein.
Fig. 8. Access to the KpHpxA active site. (a) The structure of KpHpxA is shown (pale green, ribbon and ball and stick)
with allantoin bound (blue, ball and stick). Glu53, which may serve as a gate for the active site, is shown in ball and stick
with green carbon atoms and red oxygen atoms. (b) Cutaway surface representation of KpHpxA shown with allantoin
bound (blue stick representation). The putative gating residue Glu53 is shown in green stick representation. (b) is rotated
90° about the y-axis relative to (a).

Klebsiella pneumoniae Allantoin Racemase
455
Ser79 that is closer to Ser184) and would be
approximately 6.3 Å between thiols if cysteine
residues were used in place of serine residues. The
distances of Ser79 and Ser184 to C5 of allantoin are
3.2 and 4.2 Å, respectively. It should be noted that
these distances reflect the nature of the enol form of
the ligand; both the (R)- and (S)-enantiomers of
allantoin would be expected to bind in slightly
different orientations (see modeling below).
enantiomers of allantoin. Due to slow (knon of
2×10−4 s−1) nonenzymatic racemization of allanto-
in, the individual enantiomers of this compound are
not commercially available. However, both isomers
can be prepared enzymatically from uric acid and
racemic allantoin for (S)- and (R)-allantoin, respec-
tively (see Materials and Methods).
The difference in the CD spectra of the allantoin
enantiomers provides a means to assay the
racemase activity of KpHpxA. Figure 5a shows
the change in CD spectra with respect to time for
the KpHpxA conversion of either (S)-allantoin or
(R)-allantoin to the racemic mixture. Time courses
of the reactions above are provided in Fig. 5b.
KpHpxA catalyzes the racemization effectively,
with a kcat/Km of 6.0×105 M−1 s−1 and a rate
enhancement (kcat/knon) of 1.2×106.
As a result of nonenzymatic decay of HIU and
OHCU and slow racemization of allantoin, the
efficient and economical catabolism of uric acid by
a pathway such as that used by Klebsiella sp. requires
a highly effective allantoin racemase. A high level of
allantoin to allantoate throughput would necessitate
a racemase with an efficiency that is equal or greater
than that of the associated allantoinase enzyme.
Although there are no data available for the
Klebsiella sp. allantoinase, the catalytic efficiency of
KpHpxA (kcat/Km of 6.0×105 M−1 s−1) is signifi-
cantly higher than that of the metal-dependent and
metal-independent allantoinase enzymes (kcat/Km of
7000 M−1 s−1 for Phaseolus vulgaris,31 24,000 M−1 s−1
for Escherichia coli11 and 48,000 M− 1 s− 1 for
Pseudomonas fluorescens12).
Members of the aspartate/glutamate racemase
superfamily are known to employ conserved cyste-
ines for catalysis of racemization.18,19 Multiple
sequence alignments of several aspartate/glutamate
racemase members with KpHpxA (Fig. 7) indicate
that the catalytic residues in KpHpxA are Cys79 and
Cys184. To probe the importance of these residues
for KpHpxA activity, we constructed the C79S,
C184S and C79S/C184S KpHpxA mutants and
tested their ability to racemize (R)- or (S)-allantoin.
Kinetic analysis by CD revealed that mutation of
either of the conserved cysteine residues to serine
totally inactivated the enzyme. This result is similar
to the observations for hydantoin racemase from
Sinorhizobium meliloti20 and is consistent with the
two-base mechanism that has been proposed for
other members of this family.
An important structural element observed in the
liganded KpHpxA structures is the presence of a
putative oxyanion hole. This pocket, created by the
backbone amide nitrogens of Phe80 and Gly185
(Fig. 3), which also serves to orient the hydantoin
ring by interacting with the C4 oxygen atom, could
stabilize the negative charge that builds up at this
oxygen atom along the reaction coordinate. This
interaction is observed in both the allantoin (Fig.
3a) and 5-acetylhydantoin (Fig. 3b) complex struc-
tures, indicating its importance for ligand binding.
In addition, the enol form of allantoin bound in the
active site provides evidence that this pocket may
serve to stabilize an oxyanion because this ligand is
structurally similar to the proposed enolate inter-
mediate (see discussion of mechanism below).
One of the observed differences between the
structures of KpHpxA and hydantoin racemase is
the presence of a polar region in the binding pocket
of KpHpxA populated by several threonine side
chains. This difference presumably stems from the
dissimilarity in the substituent at the 5-position of
the hydantoin ring. While hydantoin racemase
accommodates nonpolar, aliphatic and aromatic
groups such as ethyl, isobutyl and benzyl, the ureido
group at the 5-position of allantoin would require a
more hydrophilic environment. This threonine-rich
pocket is also likely to be an important determinant
of selectivity. An examination of the two liganded
structures of KpHpxA (Fig. 3) indicates that this
region provides an anchoring point for the tail region
of the ligand and aids in the orientation of the
hydantoin ring by securing the 5-substituent in this
pocket. The observation of selectivity for the (R)-
enantiomer of 5-acetylhydantoin is likely a result of
the interactions at this threonine pocket. Binding of
the acetyl group of (S)-5-acetylhydantoin in this
pocket would drive the hydantoin ring to a position
almost perpendicular to that observed in the
structure and would result in several unfavorable
protein–ligand interactions.
Catalysis of allantoin racemization by KpHpxA
Modeling of substrates in KpHpxA active site
Based upon sequence similarity to members of the
aspartate/glutamate racemase superfamily,
KpHpxA enzyme is annotated as an allantoin
racemase (GenBank accession no. ACG63342.1).2
To verify this assignment of function, we tested the
ability of KpHpxA to racemize the (R)- and (S)-
To better understand the nature of KpHpxA
active-site interactions with the substrates, we
performed a simple docking experiment. Both of
the (R)- and (S)-isomers of allantoin were manually
placed in the active site of KpHpxA and energy
minimized while keeping the positions of the

456
Klebsiella pneumoniae Allantoin Racemase
protein side chains fixed. The results (Fig. 6)
illustrate that the two active-site cysteine residues
are within a reasonable distance of allantoin C5 to
participate in catalysis of the racemization reaction.
In addition, the observed orientations of both
isomers are consistent with our assertion that ligand
binding is dominated by two main interactions, the
carbonyl of C4 in the oxyanion hole and the contacts
made by the ureido tail with the threonine pocket of
the KpHpxA active site.
species drives the attack of one of the thiols, leading to
product formation or regeneration of the substrate.
Product release is likely driven by the conformational
change that the ligand undergoes after reprotonation.
The change from sp2 to sp3 hybridization at C4 also
causes a shift in the ureido tail, bringing the –NH
group into an unfavorable position relative to the thiol
of the deprotonating cysteine. This clash likely leads
to movement of Cys79 and Glu53 away from the
departing ligand, which opens the active site and
allows for product release to occur.
Recent work on glutamate racemase has implicat-
ed additional residues in the active site that
participate in catalysis through their interactions
with the catalytic cysteine residues.18,30 The pres-
ence of these acidic residues was used to explain
why the cysteine-to-serine mutants retained some
level of racemase activity.30,34 No such acidic
residues are observed within hydrogen-bonding
distance of either of the catalytic cysteines in the
structure of KpHpxA. This is consistent with the
total loss of activity observed in both of the C79S
and C184S mutants.
Mechanistic implications
While there are no mechanistic studies reported
on allantoin racemase, insights can be gleaned from
the structural and kinetic data reported herein and
results of studies on other aspartate/glutamate
racemase superfamily members.18–20,30,32,33 Despite
the observed asymmetry in the active site, KpHpxA
catalyzes racemization of both allantoin isomers
with approximately the same affinity and rate. The
structures of liganded KpHpxA reveal a polar
threonine pocket that interacts with the ureido tail
group of allantoin and serves to orient the hydantoin
ring. The other main interaction with allantoin
occurs through the C4 carbonyl oxygen atom and,
while mediating the lateral motion (in reference to
the plane of the ring), does not preclude rotation of
the molecule. These anchor points, therefore, allow
for optimal positioning of either of the two isomers
with respect to the two active-site cysteines (Fig. 8).
Based upon the data presented herein and the
known chemistry of hydantoin racemases, a mecha-
nism for the KpHpxA-catalyzed allantoin racemase
reaction is proposed (Scheme 2). Analogous to the
two-base mechanism proposed for other members of
this family, the two active-site cysteine residues,
Cys79 and Cys184, catalyze the racemization reaction
by acting to both deprotonate and reprotonate
allantoin at the C4 position. Deprotonation of either
isomer leads to an enolate intermediate, with stabili-
zation of the negative charge provided by the
oxyanion hole. Collapse of the reactive oxyanion
Conclusions
In this paper, we have reported the first structural
characterization of an allantoin racemase. The
structures presented reveal several important fea-
tures of the active site of this enzyme, including an
oxyanion hole and a polar binding region that
strongly interacts with the ureido tail of allantoin. In
addition, measurement of the kinetic parameters by
CD demonstrates that KpHpxA catalyzes the
racemization of allantoin, enhancing the rate 6
orders of magnitude over the nonenzymatic rate.
This structural and biochemical characterization of
K. pneumonia allantoin racemase expands the
breadth of the aspartate/glutamate racemase super-
family and provides additional support for the two-
base mechanism employed by this class of cofactor-
independent racemases.
Scheme 2. Proposed mechanism of catalysis for racemization of allantoin by KpHpxA.

Klebsiella pneumoniae Allantoin Racemase
457
Data collection, processing, structure determination
and refinement
Materials and Methods
Protein purification and crystallization
The SeMet data were collected at 100 K at the
Northeastern Collaborative Access Team (NE-CAT)
beamline 24-ID-C of the Advanced Photon Source at
Argonne National Laboratory at the maximum f″ for
selenium as determined by a fluorescence scan of the
crystal. The SeMet data set was collected over 360° using a
1° oscillation range. The space group was determined to
be P6322 with two molecules per asymmetric unit
corresponding to a solvent content of 51%. Data were
indexed and scaled using HKL2000,35 and heavy-atom
sites were located using the SHELX suite.36 The program
autoSHARP37 was used for refinement of heavy-atom
positions, phasing, calculation of residual maps, density
modification and automated model building. The pro-
gram was able to correctly place 469 out of 494 residues in
two chains with the correct side chains. Additional
residues and water molecules were placed during iterative
rounds of manual model building using Coot38 followed
by refinement using Refmac5.39
The data for ligand-bound complexes were collected
at the NE-CAT beamlines 24-ID-C and 24-ID-E at a
wavelength of 0.979 Å. These data also indexed in space
group P6322 and had similar unit cell dimensions.
Refinements were carried out as detailed above using
the SeMet structure as a starting point. The ligands were
built into difference density only after the refinement
had converged. Data collection statistics are summarized
in Table 1, and refinement statistics are summarized in
Table 2.
The gene encoding KpHpxA was cloned from
genomic DNA from K. pneumoniae subsp. pneumoniae
(Schroeter) Trevisan MGH78578 (American Type Culture
Collection 700721) and placed in a modified pET28
(Novagen) vector encoding an N-terminal 6-His tag by
standard molecular biology techniques. The C79S, C184S
and C79S/C184S mutant plasmids were made at the
Cornell Protein Production and Purification Facility
using site-directed mutagenesis of the native gene.
Briefly, mutagenesis was performed on the hpxA gene
by a standard PCR protocol using PfuTurbo DNA
polymerase (Invitrogen) and DpnI (New England
Biolabs) to digest the methylated parental DNA prior
to transformation. The presence of the mutated residues
was verified by sequencing.
For protein expression in all cases, the plasmid was
transformed into B834(DE3) (methionine auxotrophic)
cells, and the protein was expressed and purified as
follows. For SeMet protein, the cells were grown in M9
minimal media supplemented with 0.4% glucose, minimal
essential vitamin solution (Invitrogen), 2 mM MgSO4,
90 mM FeSO4 (25 mg/L), 100 μM CaCl2, 40 mg/L each of
19 amino acids (methionine excluded), 50 mg/L of
seleno-L-methionine and 50 mg/L kanamycin. The cells
were grown at 37 °C to an OD600 of 0.5 before reducing
the temperature to 15 °C. After 30 min at this
temperature, the cells were induced with 0.5 mM
isopropyl-β,D-thiogalactopyranoside and grown for an
additional 16 h. The cells were harvested by centrifu-
gation, and the pellet was stored at −20 °C prior to
purification. For native protein, the procedure followed
was the same with the exception that lysogeny broth
was used as a growth medium.
Measurement of racemization activity and
steady-state kinetics
KpHpxA racemization activity was calculated from
changes in CD spectra over time. All reactions were
carried out in buffer containing 10 mM NaCl and 10 mM
Hepes, pH 7.5. The enzyme tested for activity was
exchanged into the buffer above prior to analysis. (R)-
and (S)-allantoin samples were prepared as follows
immediately prior to measurement of rates. For (S)-
allantoin, 20 μmol of uric acid in 10 mM NaCl and
10 mM Hepes, pH 7.5, was incubated with 2 units of
uricase from Candida sp. (Sigma), 5 μg of K. pneumoniae
HIU hydrolase2 and 5 μg of K. pneumoniae OHCU
decarboxylase29 for 30 min at 25 °C. The stereospecific
production of (S)-allantoin was monitored by UV at
260 nm,29 and the final concentration was confirmed by
comparison to a standard curve constructed using racemic
allantoin. Upon completion of the reaction, the enzymes
were removed by filtration through a 10-kDa-cutoff
centrifugal concentrator.
(R)-allantoin was produced by incubating 40 μmol of
racemic allantoin with 10 μg of E. coli allantoinase10 for
20 min at 25 °C. The progress from racemic to pure (R)-
allantoin was monitored by CD. Upon completion of the
reaction, the enzyme was removed by filtration through a
10-kDa-cutoff centrifugal concentrator. Since inhibition of
KpHpxA by allantoate was not observed to occur (data not
shown), the resulting mixture of (R)-allantoin and allanto-
ate was used without further treatment.
For protein purification, the pellets were thawed in
buffer containing 300 mM NaCl, 10 mM imidazole,
2
mM dithiothreitol (DTT) and 50 mM sodium
phosphate, pH 7.6. The cells were lysed by sonication
and centrifuged to remove cellular debris. The cleared
lysate was passed through a column containing pre-
equilibrated Ni-NTA resin and then washed with a
similar buffer containing 20 mM imidazole and 10%
glycerol. The protein was eluted with buffer containing
300 mM NaCl, 2 mM DTT and 250 mM imidazole,
pH 7.6, and immediately buffer exchanged into 30 mM
NaCl, 2 mM DTT and 10 mM sodium acetate, pH 5.6.
The protein was concentrated to 15 mg/mL using a
Centricon centrifugal concentrator (Amicon) and deter-
mined to be greater than 95% pure by SDS-PAGE
electrophoresis. Aliquots of the protein were flash
frozen immediately after buffer exchange and stored
at −80 °C for crystallization trials. Crystallization was
performed by the hanging-drop vapor diffusion method
at 18 °C. For both the native and SeMet proteins,
crystals formed after 3–5 days in 24–34% polyethylene
glycol 400 with 0.1 M sodium acetate, pH 3.7–4.1.
Crystals were harvested and frozen without additional
cryoprotectant. For allantoin and 5-acetylhydantoin
complexes, crystals were soaked for 2 h in solutions
of mother liquor supplemented with 5 mM ligand.

458
Klebsiella pneumoniae Allantoin Racemase
Table 1. Data collection statistics
KpHpxA
C79S/C184S–allantoin
KpHpxA
C79S/C184S–5-acetylhydantion
KpHpxA-SeMet
Resolution (Å)
Wavelength (Å)
Beamline
Space group
a (Å)
c (Å)
50.0–2.1
0.9792
24-ID-C
P6322
124.8
126.8
50.0–2.0
0.9791
24-ID-E
P6322
124.9
127.0
50.0–1.82
0.9791
24-ID-E
P6322
125.0
127.0
No. of reflections
Unique reflections
Average I/σ
Redundancy
Completeness (%)
Rsyma (%)
106,706
28,561
15.0 (2.1)
3.7 (3.5)
94.6 (99.6)
9.7 (40.1)
197,739
39,452
25.4 (4.3)
5.0 (4.5)
98.3 (98.2)
9.6 (38.1)
805,670
52,509
47.0 (7.9)
15.3 (11.3)
99.9 (100.0)
7.0 (30.1)
Numbers in parentheses correspond to the highest-resolution shell.
a
Rsym =∑∑I|Ii −〈I〉|/∑〈I〉, where 〈I〉 is the mean intensity of the N reflections with intensities Ii and common indices h, k and l.
CD spectra and kinetics were collected on an AVIV
Biomedical (Lakewood, NJ) CD spectrometer, Model
202-01. The spectra were collected at 25 °C from 190 to
260 nm with a 1-nm step size, a 1-nm bandwidth and a
1-mm cell. Initial rates of reactions were calculated at
various substrate concentrations and were taken from
the first 10 s of the reaction (reaction less than 10%
complete). The signals at 208 and 213 nm, respectively,
were monitored for the (S)- and (R)-reactions. The
kinetic parameters were calculated from a fit of the
initial rates by the Michaelis–Menten equation using
KaleidaGraph (Synergy Software).
the enol form of allantoin from the C79S/C184S KpHpxA–
allantoin structure. The protein was truncated to a shell
containing all atoms within 20 Å of the ligand. Water
molecules were removed, and the protein preparation
utility was used to add hydrogens and to ensure proper
ionic states of amino acid side chains. All of the protein
atoms were frozen in place, and only the allantoin ligand
was allowed to move during minimization. The calcula-
tions were completed using the AMBER⁎ force field42,43
with a distance-dependent dielectric that was further
attenuated by a factor of 4. The energy minimization relied
upon the truncated Newton conjugate gradient
technique44 and was considered to have converged when
the energy gradient was less than 0.01 kJ/mol.
The topology diagram was based upon the results from
the PDBsum server.45 The sequence alignment was carried
out using Clustal W,25 and the corresponding figure was
made using the ESPript server.26 The Dali server21 was
used to search for structural homologues of KpHpxA. All
other figures were made using PyMOL46 and ChemBio-
Draw (CambridgeSoft).
Docking of allantoin isomers in active site,
sequence alignment and structural homology search
Version 9.7.211 of the molecular modeling program
Macromodel40,41 was used to dock the (R)- and (S)-
allantoin isomers in the active site of KpHpxA. The starting
position of allantoin was derived from a superposition to
Table 2. Data refinement statistics
KpHpxA
C79S/C184S–allantoin
KpHpxA
C79S/C184S–5-acetylhydantion
KpHpxA-SeMet
Resolution (Å)
50.0–2.1
3542
50.0–2.0
3573
50.0–1.82
3518
No. of protein atoms
No. of ligand atoms
No. of water atoms
No. of reflections in working set
No. of reflections in test set
R-factora (%)
0
14
22
190
302
446
32,766
1740 (5%)
20.5
37,463
1974 (5%)
22.2
49,799
2675 (5%)
20.8
Rfreeb (%)
23.9
24.6
22.2
RMSD bonds (Å)
RMSD angles (°)
0.007
1.013
32.9
0.005
0.927
31.6
0.005
0.927
19.9
Mean B-factor (Å2)
Ramachandran plot (%)
Most favored
94.3
5.7
0.0
0.0
95.2
4.8
0.0
0.0
95.2
4.8
0.0
0.0
Additionally allowed
Generously allowed
Disallowed
a
R-factor=∑hkl||Fobs|−k|Fcalc||/∑hkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively.
For Rfree, the sum is extended over a subset of reflections (5%) excluded from all stages of refinement.
b

Klebsiella pneumoniae Allantoin Racemase
459
Accession numbers
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Acknowledgements
This work was supported by National Institutes
of Health grant GM73220 to S.E.E. We thank the
staff at the NE-CAT of the Advanced Photon
Source, Argonne National Laboratory, for advice
with data collection and processing. Data collection
at the NE-CAT beamlines was supported by award
RR-15301 from the National Center for Research
Resources at the National Institutes of Health. Use
of the Advanced Photon Source was supported by
the U.S. Department of Energy, Office of Basic
Energy Sciences, under contract number DE-AC02-
06CH11357. We acknowledge Dr. Cynthia Kinsland
of the Protein Production and Purification Facility
in the Department of Chemistry and Chemical
Biology for help with molecular biology and Ms.
Leslie Kinsland for help with manuscript prepara-
tion. J.B.F. would like to acknowledge the Tri-
Institutional Training Program in Chemical Biology
for financial support.
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Supplementary data associated with this article
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j.jmb.2011.05.016
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