Biochemical and structural characterization of a ureidoglycine aminotransferase in the klebsiella pneumoniae uric acid catabolic pathway

Article abstract of DOI:10.1021/bi1006755

Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiella pneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an α-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

Full text of DOI:10.1021/bi1006755

Biochemistry 2010, 49, 5975–5977 5975
DOI: 10.1021/bi1006755
Biochemical and Structural Characterization of a Ureidoglycine Aminotransferase
†,‡
in the Klebsiella pneumoniae Uric Acid Catabolic Pathway
Jarrod B. French and Steven E. Ealick*
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301
Received April 30, 2010; Revised Manuscript Received June 15, 2010
ABSTRACT: Many plants, fungi, and bacteria catabolize
Scheme 1: Uric Acid Catabolic Pathway in Klebsiella pneumo-
niae
a
allantoin as a mechanism for nitrogen assimilation. Recent
reports have shown that in plants and some bacteria the
product of hydrolysis of allantoin by allantoinase is the
unstable intermediate ureidoglycine. While this molecule
can spontaneously decay, genetic analysis of some bacterial
genomes indicates that an aminotransferase may be present
in the pathway. Here we present evidence that Klebsiella
pneumoniae HpxJ is an aminotransferase that preferentially
converts ureidoglycine and an R-keto acid into oxalurate
and the corresponding amino acid. We determined the
crystal structure of HpxJ, allowing us to present an
explanation for substrate specificity.
The uric acid catabolic pathway is the final stage of purine
catabolism and functions in plants and some bacteria to provide a
source of nitrogen, particularly when other nitrogen sources are
depleted (1, 2). Uric acid catabolism begins with the oxidation of
uric acid by uricase followed by ring opening and decarboxyla-
tion reactions catalyzed by 5-hydroxyisourate hydrolase and
a
The enzyme names are taken from the K. pneumoniae degrada-
tive pathway. The dashed line indicates an alternative route that
operates in other organisms.
2
-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase,
can be oxidized to oxalurate which can be used in subsequent
reactions to generate ATP and ammonia (1, 10).
respectively (3-6). The latter two steps lead to the production of
allantoin, a process that also occurs nonenzymatically after
oxidation of uric acid (1, 4). Allantoin can then be further
degraded to allantoate by an allantoinase enzyme (1).
The further catabolism of ureides follows one of two pathways.
The degradation of allantoate is catalyzed by either allantoicase
or allantoate amidohydrolase. The former produces urea and
ureidoglycolate, both of which require further catabolism for full
nitrogen assimilation. The latter pathway, which is found in
plants and some bacteria (1, 2), also results in ureidoglycolate but
has been demonstrated to proceed via ureidoglycine, a molecule
now known to be the product of the allantoate amidohydrolase-
catalyzed reaction (7).
Characterization of the fate of ureidoglycine has been compli-
cated by the fact that it is unstable in aqueous environments and
is subject to rapid nonenzymatic decay (7, 8). A recent report by
Werner et al., however, demonstrated that ureidoglycine can be
hydrolyzed by ureidoglycine aminohydrolase (9). The resulting
ureidoglycolate can be consumed by ureidoglycolate amidohydro-
lase or the urea-releasing ureidoglycolate hydrolase (ureidoglyco-
late lyase) (2, 9). Alternatively, in some organisms, ureidoglycolate
Two recent genetic studies have identified a gene cluster
encoding a purine degradative pathway in Klebsiella sp. that
proceeds through intermediates similar to those outlined above
but does so using different catalytic strategies (11, 12). Scheme 1
outlines the proposed enzymes and intermediates in this path-
way (7, 9, 12). One surprising finding in the gene cluster encod-
ing these enzymes was the presence of a gene for a protein that
0
belongs to the pyridoxal 5 -phosphate-dependent aspartate
aminotransferase superfamily. An examination of the intermedi-
ates along the degradative pathway reveals that only ureidogly-
cine has the requisite amino group needed to participate in
an amino transfer reaction. Also notable is the absence of
an enzyme capable of oxidizing ureidoglycolate to oxalurate.
Taken together, this suggests that the pathway could proceed
from ureidoglycine to oxalurate directly in some organisms. The
presence of an aminotransferase associated with purine degrada-
tion is not unique to Klebsiella sp. In a recent cross-organism
protein association analysis, a gene encoding an aminotransfer-
ase was found linked to allantoate amidohydrolase in several
organisms, particularly those lacking a ureidoglycine aminohy-
drolase (9).
†
This work was supported by National Institutes of Health Grant
To determine if a ureidoglycine aminotransferase participates
in the purine degradative pathway, we have biochemically and
structurally characterized the K. pneumoniae enzyme with this
putative function. To establish if HpxJ had aminotransferase
activity, we first examined its ability to catalyze the transfers of an
GM73220. J.B.F. acknowledges funding from the Tri-Institutional
Program in Chemical Biology.
‡
The coordinates for HpxJ have been deposited in the Protein Data
Bank under accession number 3NNK.
*To whom correspondence should be addressed. Phone: (607) 255-
7961. Fax: (607) 255-1227. E-mail: see3@cornell.edu.
r 2010 American Chemical Society
Published on Web 06/21/2010
pubs.acs.org/Biochemistry

5
976 Biochemistry, Vol. 49, No. 29, 2010
French and Ealick
amino group between aspartate and glyoxylate. To quantitate the
amino acid content in the reaction mixtures, we treated the
1
mixtures with DABS-Cl prior to HPLC analysis. Figure S1 of
the Supporting Information shows HPLC chromatograms of the
amino transfer reaction in the presence and absence of HpxJ. A
clear peak that coelutes with the glycine standard appears in the
enzyme-catalyzed reaction, while the aspartate peak diminishes
in size. The production of glycine was enzyme-dependent and
also required the presence of an amino donor.
Having established that HpxJ was capable of catalyzing amino
transfer, we then wished to determine if ureidoglycine would be
a substrate for this enzyme. Because this molecule is known to be
unstable in solution, we employed Escherichia coli allantoate
amidohydrolase (AAH) to synthesize ureidoglycine in situ. This
enzyme and the chemistry of the AAH reaction have been
recently characterized (7). The production of ureidoglycine by
the AAH reaction can be followed by monitoring the produc-
tion of ammonia with a glutamate dehydrogenase coupled assay
(
Figure S4 of the Supporting Information).
The amino acid products of the HpxJ-catalyzed amino transfer
reaction between ureidoglycine and pyruvate were followed by
HPLC after derivatization with DABS-Cl. A peak corresponding
to alanine was observed in the presence of the enzyme when
pyruvate was the amino acceptor (Figure 1A, bottom panel). We
also observed a small amount of glycine being produced both in
the presence and in the absence of added pyruvate (Figure 1A,
bottom two panels). This can be explained by the nonenzymatic
decay of ureidoglycine. The decay of this molecule produces
glyoxylate (7, 8) which can then be used as a substrate for the
reaction. HPLC traces of standards and additional amino
transfer reactions are provided as Supporting Information.
The products of the amino transfer reaction between ureido-
glycine and an R-keto acid are an amino acid and oxalurate. For
further evidence that HpxJ catalyzes the amino transfer reac-
tion, we derivatized the keto acid products of the reaction with
o-phenylenediamine and followed them by HPLC. Figure 1B
shows that oxalurate is produced in the presence of either
pyruvate (Figure 1B, third panel) or oxaloacetate (Figure 1B,
fourth panel) as the amino acceptor. As with the amino products
of the reaction, a small oxalurate peak was also observed in the
absence of added amino acceptor (Figure 1B, second panel).
One of the conserved features of the aspartate aminotransfer-
ase family is the dependence upon the cofactor PLP. The
absorbance spectrum of HpxJ (Figure 1C) is characteristic of
enzymes with bound PLP in the imine form. Treatment of this
enzyme with sodium borohydride causes the loss of the peak at
F
IGURE 1: Characterization of K. pneumoniae HpxJ. (A) HPLC
traces of the amino products of the HpxJ amino transfer reactions.
From top to bottom: controlreactionrun withHpxJin the absence of
ureidoglycine, control reaction run in the absence of HpxJ with all
other components present, reaction with HpxJ and ureidoglycine in
the absence of an exogenous amino acceptor, and HpxJ-catalyzed
reactionrun inthe presence ofureidoglycine and pyruvate. (B) HPLC
traces of the keto acid products of the HpxJ amino transfer reaction.
From top to bottom: control in the absence of HpxJ, reaction with
HpxJ and ureidoglycine in the absence of exogenous amino acceptor,
transfer reaction in the presence of pyruvate, and transfer reaction in
the presence of oxaloacetate. Note that any unreacted allantoate in
the reaction is hydrolyzed to glyoxylate during the derivatization
workup. (C) UV absorbance spectra of native HpxJ (;) and reduced
HpxJ (---). (D) Pre-steady state kinetics of the first half-reaction
catalyzed by HpxJ and rates from a fit to an exponential function.
Note that the alanine and ureidoglycine traces have been shifted
down the y-axis for the sake of clarity.
420 nm and a shift of the 333 nm peak to a slightly lower
wavelength. This is consistent with the reduction of the PLP-
imine to the amine form, a phenomenon that is also observed in
the first half of the transfer reaction as PLP is converted to
PMP (13). We also observed that this reduced form of HpxJ was
unable to catalyze the aminotransferase reaction for any of the
amino donor/acceptor pairs that we tried (data not shown).
Aminotransferases are known to be promiscuous with regard
to the identity of amino donors and acceptors (14, 15). To
examine the specificity of HpxJ for different amino acids, we
measured the pre-steady state rate for the first half-reaction of the
transfer by monitoring the conversion of PLP to PMP. The
curves and calculated rates for the different amino acids are
shown in Figure 1D. It is clear from these data that ureidoglycine
is the favored substrate, turning over 1 order of magnitude faster
than the next fastest substrate, aspartate.
To further examine this novel aminotransferase and its pre-
ference for ureidoglycine, we crystallized and determined the
structure of HpxJ using molecular replacement with Protein Data
Bank entry 1VJ0 (16) as a search model. Details about the
structure solution and model building, including collection and
refinement statistics, can be found in the Supporting Informa-
tion. The crystal structure of HpxJ showed a homotetramer as
the biologically relevant unit (Figure S2 of the Supporting
Information), an observation that was verified by size exclusion
1
Abbreviations: PLP, pyridoxal 5-phosphate; PMP, pyridoxamine
phosphate; DABS-Cl, dimethylaminobenzenesulfonyl chloride; OPD,
o-phenylenediamine; AAH, allantoate amidohydrolase; UGly, ureido-
glycine.

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Biochemistry, Vol. 49, No. 29, 2010 5977
converged to a similar orientation of the ligand in the active site.
The structure of the HpxJ active site with the energy-minimized
ligand bound is shown in Figure 2B. What is apparent from this
model is that, in addition to interactions with the amino and
acid groups made by the PLP molecule, backbone atoms of a
conserved proline-proline pair (Pro19 and Pro20), and a con-
served arginine (Arg360), the ureidoglycine tail makes several
hydrogen bond contacts with Asn264 and Gln39. These amino
acids, which replace bulkier tyrosine and histidine residues seen in
other aminotransferases, not only allow space for the larger
substrate but also appear to directly interact with the substrate.
In summary, we have confirmed that HpxJ is an aminotrans-
ferase that catalyzes a novel transfer reaction between ureidogly-
cine and an R-keto acid. We have provided biochemical and
structural evidence of this enzyme’s specificity for ureidoglycine.
Our data provide an explanation for the presence of this gene in
the K. pneumoniae Hpx gene cluster and elucidate a novel branch
in the ureide catabolic pathway.
ACKNOWLEDGMENT
F
IGURE 2: Active site of HpxJ. (A) Structure of the PLP binding site
of HpxJ showing a molecule of PLP covalently bound to lysine 200.
- F density is shown contoured at 3σ around the PLP and lysine
We acknowledge Dr. Cynthia Kinsland at the Cornell Pro-
tein Production Facility for assistance with molecular biology,
Kenneth Gee for assistance with HPLC experiments, and Leslie
Kinsland for assistance with the preparation of the manuscript.
We also thank the NE-CAT staff at beamline 24-ID-C of the
Advanced Photon Source for assistance with data collection.
F
O
C
residue. (B) Superposition of the activesiteofHpxJ(green) and Aedes
aegypti alanine glyoxylate aminotransferase (gray). Ureidoglycine is
shown docked in the active site (yellow, ball and stick).
chromatography. Because this enzyme was highly similar to other
PLP-dependent aminotransferases, it was not surprising to find a
covalently bound PLP molecule in the active site (Figure 2A).
The electron density allowed for the unambiguous placement of
the PLP in an orientation that superimposed well with PLP in
homologous structures.
SUPPORTING INFORMATION AVAILABLE
Experimental details, data collection and refinement statistics,
and additional figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
While the members of the alanine-glyoxylate aminotransferase
superfamily are highly structurally conserved, there is a relatively
large degree of variation among active site residues. With the
exception of the highly conserved amino acids that interact with
the PLP molecule, differences in active site residues account for
the observed differences in substrate selectivity (see Figure S3 of
the Supporting Information for a sequence alignment). A super-
position of HpxJ with structurally homologous aminotransferases
shows several obvious differences in the active sites of these pro-
teins. The most notable of these is an asparagine (Asn264) in place
of a tyrosine residue and a glutamine (Gln39) in place of a histi-
dine. The side chains of these residues point into the active site and
are well positioned to make contact with incoming substrates.
To improve our understanding of the selectivity of HpxJ for
ureidoglycine over other amino acids, we modeled the ligand into
the active site of our structure. Despite starting from several
different conformers of ureidoglycine, all of the simulations
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