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The biosynthesis of purines requires amino acid precursors and the investment of ATP. In contrast, most organisms appear unable to obtain energy or useful compounds from purine

catabolism. In general, no energy is produced by the purine break-down, and the compounds formed are often discarded as waste material1,2. In some cases, such as in apes, birds and certain reptiles, uric acid, which retains all the carbon and nitrogen atoms of the purine ring, is discarded. When the degradation pathway is com-pleted, yielding CO2, NH3 and glyoxylate as end products, as occurs in plants, the purpose seems to be the recovery of the purine ring nitrogen3–5. Organisms exist, however, that use purines as a nutri-ent and are able to grow with oxidized purines as the only source of nitrogen, carbon and energy2. The reasons for an efficient use of purines are not completely understood but are probably related to an efficient use of glyoxylate. This end product of the purine breakdown interferes with respiration6 and imposes metabolic problems on aerobic organisms, which have a detoxification sys-tem for this compound. Two vertebrate genes, AGXT1 and AGXT2, encode vitamin B6–dependent enzymes with alanine-glyoxylate aminotransferase activity that are targeted to peroxisomes and to mitochondria, respectively. In humans, defects in the peroxisomal protein (AGXT1) cause accumulation of a glyoxylate oxidation product, as observed in the genetic disease primary oxaluria7.

A bacterial gene related to alanine-glyoxylate aminotransferase was first noticed in the purine degradation cluster of Bacillus subtilis and named pucG8. Among bacteria, Bacillus species are known to possess an efficient metabolic system for the use of oxidized purines. As an extreme example, Bacillus fastidiosus grows only on medium containing urate, allantoin or allantoate and only germinates on medium containing urate9. The more common B. subtilis, a faculta-tive aerobe found in soil and in the animal gut10,11, does not have the fastidious growth requirements of its relative but is still able to derive nitrogen, carbon and energy from purines2,8.

By combining previous genetic and biochemical information with genome comparison and logic analysis, we identify here the product

of the allantoate amidohydrolase reaction, (S)-ureidoglycine, as the candidate amino group donor for the transamination catalyzed by the PucG protein. We provide NMR and crystallographic evidence that pucG encodes a vitamin B6–dependent enzyme with UGXT activity. This activity prevents glyoxylate accumulation and enables the recovery of glycine from the purine breakdown with no energy cost. In the UGXT-catalyzed reaction, a single substrate provides both the amino group donor and the amino group acceptor, illustrat-ing a new example of a transamination sequence. Structural similar-ity between the UGXT and AGXT1 proteins suggests the existence of a functional link between purine catabolism and the glyoxylate detoxification system of metazoa.

RESULTSAn aminotransferase involved in ureidoglycine metabolismOpening of the purine ring in the purine degradation pathway yields allantoate, and hydrolysis of allantoate by allantoate amidohydrolase (AAH) produces (S)-ureidoglycine, an unstable α-amino acid (Fig. 1a). Plants and some bacteria possess an enzyme (UGHY) catalyzing hydro-lysis of the AAH reaction product into ureidoglycolate4,5. However, UGHY-encoding genes (ughy) are not present in some bacteria, such as B. subtilis, that possess AAH-encoding genes (allC). Comparison of purine degradation clusters in various bacteria (Fig. 1b) shows that allC is alternatively associated with two unrelated genes: ughy or pucG. This kind of gene association (allC implies ughy or pucG) can be interpreted biochemically as ughy and pucG being unrelated genes encoding pro-teins with the same catalytic activity or with the presence of a branch point in the metabolic pathway12,13. The similarity of PucG to alanine-glyoxylate aminotransferase favors the hypothesis that the protein acts as a transaminase for the production of glycine4, suggesting that pucG encodes (S)-ureidoglycine–glyoxylate aminotransferase.

PucG is a (S)-ureidoglycine–glyoxylate aminotransferasePucG was cloned from B. subtilis, and the untagged recombi-nant protein was overproduced in Escherichia coli and purified to

1Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy. 2Department of Biological Chemistry, University of Padua, Padua, Italy. 3venetian Institute of Molecular Medicine, Padua, Italy. 4These authors contributed equally to this work. 5Present address: Department of Biological Chemistry, University of Padua, Padua, Italy. *e-mail: riccardo.percudani@unipr.it

an aminotransferase branch point connects purine catabolism to amino acid recyclingIleana ramazzina1,4, roberto Costa1,4,5, Laura Cendron2,3, rodolfo Berni1, alessio peracchi1, Giuseppe Zanotti2,3 & riccardo percudani1*

Although amino acids are known precursors of purines, a pathway for the direct recycling of amino acids from purines has never been described at the molecular level. We provide NMR and crystallographic evidence that the PucG protein from Bacillus subtilis catalyzes the transamination between an unstable intermediate ((S)-ureidoglycine) and the end product of purine catabolism (glyoxylate) to yield oxalurate and glycine. This activity enables soil and gut bacteria to use the animal purine waste as a source of carbon and nitrogen. The reaction catalyzed by (S)-ureidoglycine–glyoxylate aminotransferase (UGXT) illustrates a transamination sequence in which the same substrate provides both the amino group donor and, via its spontaneous decay, the amino group acceptor. Structural comparison and mutational analysis suggest a molecular rationale for the functional divergence between UGXT and peroxisomal alanine-glyoxylate aminotransferase, a fundamental enzyme for glyoxylate detoxification in humans.

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apparent homogeneity by gel filtration and ion exchange chromato-graphy (Supplementary Fig. 1a). The protein had an absorbance spectrum typical of enzymes containing pyridoxal phosphate (PLP; a vitamin B6 derivative), with a peak at 412 nm in addition to the peak at 280 nm (Supplementary Fig. 1b). Activity of the protein on ureidoglycine, the unstable product of the AAH reac-tion (see Fig. 1a), was assayed by 13C NMR spectroscopy using uniformly 13C-labeled allantoate and unlabeled glyoxylate in the presence of AllC. The time course of the reaction showed forma-tion of a stable product having the same 13C spectrum as oxalurate (Fig. 2a and Supplementary Fig. 2). Reactions conducted with allantoate specifically labeled at the pro-S carbon (position C2) showed incorporation of 13C into C2 of oxalurate (Supplementary Fig. 2), providing evidence that the amino donor substrate is the S enantiomer of ureidoglycine. The stereochemistry at the α-carbon in (S)-ureidoglycine is the same as that of L amino acids.

The activity of PucG on glyoxylate was assayed with un-labeled allan toate in the presence of AllC and uniformly 13C- labeled glyoxy late deriving from the spontaneous decay of labeled

(S)-ureidoglycine. The spectrum showed formation of 13C-labeled glycine as the reaction product (Supplementary Fig. 3). Formation of glycine was also observed using a coupled spectrophotometric assay with glycine oxidase (Supplementary Fig. 4). These results indicate that pucG encodes a protein with UGXT activity.

UGXT reaction in the absence of amino group acceptorsThanks to the absorption of oxalurate in the UV region, which is higher than that of the other metabolites involved in the reaction, the transamination kinetics were monitored spectrophotometrically. As ureidoglycine spontaneously releases urea and ammonia at neutral pH (Supplementary Fig. 5), yielding glyoxylate at a rate constant of ~4 × 10−4 s−1, the UGXT reaction could also occur in the absence of added amino group acceptor. In a reaction mixture containing AllC, UGXT and glyoxylate, the allantoate substrate was stoichio-metrically converted to oxalurate (Fig. 2b). Formation of oxalurate, however, was also observed when glyoxylate was omitted. In that case, the reaction proceeded, after an initial lag, with the same rate of the spontaneous decay of ureidoglycine and yielded half the amount

Scale (nt.)

0k 1k 2k 3k 4k 5k 6k 7kpucG

allC ughy

Bacillus subtilis str. 168

Bacillus clausii

Bacillus licheniformis

Staphylococcus carnosus

Bacillus sp. B14905

Enterococcus faecalis

Escherichia coli

Deinococcus radiodurans

Xanthomonas campestris

Caulobacter crescentus

Yersinia intermedia

ba

Guanine Adenine

Uric acid

(S)-Ureidoglycine

(S)-Ureidoglycolate

Glyoxylate Oxalurate

–O –O

–O

–O

–O

–O

O

NH

ONH H

NH22NH

O

Allantoate NH3, CO2

O

NH

O

2NH

3H N+

H

O

NH

OHO

HO

O H

O

NH

OO

Glycine

O

+

3H N HH

Urea

3NH , urea

AllC

UGHY

PucG

NH2

NH2

3NH

0 min

15 min

80 min

C4

C2,C7a b

C5

d

g

g

d

d

g

g

180 170 160 150 140 130 120 110 100 90 80 70 60

164 162 160 158 156

C2C4 C5

CO2 (C7)

–O O

NH

O

NH24

5 2NH H

NH2

O7

–O O

NH

O

NH2

O

4

5 2

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

Abs

orba

nce

at 2

20 n

m

00 10 20 30

Time (min)40 50

Glyoxylate

PucG

Figure 2 | Aminotransferase activity of UGXT. (a) Time course of the enzymatic conversion of uniformly labeled [15N,13C]allantoate in the presence of AllC and PucG. 13C spectra were collected at 25 °C in 0.1 M potassium phosphate, 80% D2o, pH 8.0; signals from glycerol and dioxane (66.5 p.p.m., reference) are marked by lowercase letters. (b) Formation of oxalurate by the PucG reaction monitored spectrophotometrically in the presence and in the absence of glyoxylate. The reaction was initiated by addition of AllC to a solution containing 0.3 mM allantoate.

Figure 1 | identification of pucG as UGXT. (a) Proposed pathway for the formation and degradation of (S)-ureidoglycine. (b) Comparison of the genetic context of the AAH-encoding genes (allC, blue) in various bacteria showing the alternative association with ughy (green) and pucG (red) genes.

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of oxalurate (Fig. 2b). We conclude that in the absence of an amino group acceptor, part of the ureidoglycine donates its amino group to the enzyme in a half-transamination reaction, and part of the ureidoglycine undergoes decay, producing the substrate necessary to complete the reaction according to the following stoichiometry: 2((S)- ureidoglycine) + H2O → oxalurate + glycine + NH3 + urea. Thus, in the UGXT-catalyzed reaction, the two central carbon atoms of the purine ring are recovered as glycine. These are the same carbons that are provided by glycine in the purine biosynthetic pathway.

The use of alternative amino group acceptors was assessed by taking into account the background level of transamination. Besides glyoxylate, which is the preferred substrate, UGXT was able to effi-ciently use various keto acids (Supplementary Table 1). By contrast,

none of the amino acids that are substrates for AGXT1 and related aminotransferases (Ala, Asp, Glu, Ser, Val) proved to be effective substrates for UGXT.

crystal structure of UGXTThe crystal structure of B. subtilis UGXT, determined at 2.05-Å resolution (Supplementary Table 2), shows a protein homo-dimer (Fig. 3a,b). The N-terminal residues 1–6, C-terminal resi-dues 412–418 and internal residues 225–242 could not be seen in the crystal structure. Residues 225–242 belong to an extra segment occurring in UGXT sequences but not in related amino-transferases (Supplementary Fig. 6). Each UGXT monomer consists of an extended N-terminal segment (residues 7–14), a 3-layer main domain (residues 15–291) and a C-terminal domain (residues 292–411). The N-terminal segment of one monomer wraps around the other monomer, and a large flat surface formed by two α-helices and three loops of the main domain packs against the equivalent region of the other monomer, burying a total of 2,800 Å2 surface area per monomer. The overall structure is similar to that of PLP-dependent enzymes belonging to the so-called type I–fold14.

The PLP cofactor binds at the dimer interface (Fig. 3a,b). Its electron density is continuous with that of Lys200 (Supplementary Fig. 7a), consistent with formation of a Schiff base via covalent attachment of the ε-amino group of the lysine side chain and C4′ of the PLP. The positioning of the cofactor at the active site also suggests the presence of various noncovalent interactions (Supplementary Fig. 7b–d). The PLP ring is stacked between a phenylalanine ring (Phe99) and a valine residue (Val176) and is held in place by hydrogen-bonding interactions between the exo-cyclic O3 hydroxyl of the pyridine ring and the side chain of Thr149 and between the PLP ring nitrogen and the Asp174 side chain. This latter inter action is common to all PLP-dependent enzymes of the type I–fold. Oxygen atoms of cofactor phosphate are at hydrogen-bonding distance of Thr72, Ser73, Arg74, Gln198 from the same monomer and Thr269 from the other monomer.

Binding of the unstable ureidoglycine substrate at the UGXT active site (Fig. 3c) was modeled by homology, using the known structure of AGXT-substrate complexes7,15. Only the S enantiomer of ureidoglycine could be easily fitted into the UGXT active site. In the enzyme-substrate model, the carboxyl group of ureidoglycine is neutralized through interaction with the side chain of Arg365, a residue conserved in related aminotransferases (see Supplementary Figs. 6 and 7d), while the amino group is positioned near to the C4′ atom of PLP (Fig. 3c). The ureido side chain of (S)-ureidoglycine is directed toward a region of the active site occupied by residues Gln39, Arg74, Asn266 and Thr269, suggesting, in particular, hydro-gen bond interaction between the terminal amide group of the substrate and the amide group of Asn266. Although Thr269 is con-served in related aminotransferases, Arg74, Gln39 and Asn266 were identified as distinctive UGXT residues in sequence conservation analysis (see below).

Structural comparison with other aminotransferasesThe UGXT protein has significant sequence similarity with other aminotransferases of known structure. The most similar proteins are a putative alanine-glyoxylate aminotransferase from Nostoc sp. pcc 7120 (PDB: 1VJO; 31% identity) and the peroxisomal alanine-glyox-ylate aminotransferase (AGXT1) from Homo sapiens (PDB: 1H0C; 29% identity). A lower level of similarity is found with yeast AGXT (PDB: 2BKW; 21% identity). Structural superposition of UGXT and human AGXT1 (Supplementary Fig. 8a) reveals a remarkable similarity, with an r.m.s. deviation of 1.3 Å. Nevertheless, the amino donor specificities of the two proteins are clearly distinct: UGXT showed a very low activity with alanine, whereas human AGXT1 was unable to transaminate (S)-ureidoglycine (Supplementary Table 3).

Figure 3 | UGXT structure. (a) Stereo pair of UGXT dimer Cα chains. The PlP cofactor is shown in red. The N- and C-terminal residues identified in the structure and every other 50 residues are numbered. (b) Cartoon representation of the UGXT dimer highlighting the secondary structure organization and covalent attachment of PlP to lys200. (c) Stereo view of the (S)-ureidoglycine substrate (green carbons) docked at the active site. The PlP cofactor and polar residues within 4 Å from the ligand are shown as sticks.

250 250

50 50

100 100

N

a

b

c

N

200 200

150 150350 350

300 300

C C

Lys200

NC

Arg365 Arg365

(B)Thr269

Thr269 Thr269Asn266Asn266 Arg74 Arg74

PLP PLP

(B)Gln39

(B)Gln39

(B)Thr269

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To understand the basis for the different substrate specificities, we examined residues that are conserved in UGXT sequences but are different between UGXT and AGXT1 (see Supplementary Fig. 6). This analysis revealed distinctive substitutions in residues located at the active site, at a distance of less than 8 Å from the PLP cofactor: Gln39, Arg74, Phe99, Leu102, Asn266 (Supplementary Fig. 8b). Phe99, a residue involved in stacking interaction with PLP, is substi-tuted by tryptophan in AGXT1, but a phenylalanine residue is com-monly found in other related aminotransferases (Fig. 4a). Similarly, Leu102 is found in other aminotransferases acting on alanine, such

as plant serine-glyoxylate aminotransferase (SGAT). By contrast, Gln39, Arg74 and Asn266 appear to be key residues of UGXT. Asn266 is only present in two bacterial SGAT-like sequences, and Gln39 and Arg74 are only found in UGXT (Fig. 4a).

The relative importance of the identified residues in the activity of UGXT was probed by site-directed mutagenesis. The three dis-tinctive UGXT active site residues, Gln39, Arg74 and Asn266, were individually substituted by residues found at corresponding posi-tions in AGXT (see Supplementary Fig. 6), generating the Q39H, R74H and N266Y mutants. An additional mutant at position 266 was generated with a more conservative substitution (N266S), as observed in some AGXT-related aminotransferases of unknown function16. With the exception of R74H, recombinant expression of mutants yielded soluble proteins that could be purified close to homogeneity by ion exchange chromatography. Comparison with the wild-type enzyme showed that the transamination activity on ureidoglycine was substantially reduced in Q39H and N266S and abolished in N266Y (Fig. 4b). According to the proposed enzyme-substrate model (see Fig. 3c), binding of ureidoglycine at the N266Y mutant is prevented by steric hindrance between the ureido side chain and the tyrosine ring (see also Supplementary Fig. 8b). Notably, N266Y and N266S mutants showed an increased activity in the alanine transamination reaction (Supplementary Table 3), consistent with a role for this residue in determining the specificity for the amino donor substrate.

DiScUSSioNTransamination of (S)-ureidoglycine and glyoxylate by UGXT enables the recovery of an amino acid (glycine) from purine degra-dation. The other constituents of the molecular pathway are known enzymes of the oxidative degradation of purines1,2. An anaerobic pathway for the production of glycine starting from the hydrolysis of xanthine has been proposed17, but the constituents of this path-way are completely unknown at the molecular level. Glycine can also be formed by transamination of glyoxylate and alanine by the AGXT enzyme. Because in this reaction glycine is produced at the expense of another amino acid, the AGXT activity is properly con-sidered a detoxification activity7,18. The striking structural similarity and close evolutionary relatedness between UGXT and AGXT1 (see Fig. 4a and Supplementary Fig. 8) suggest that the original func-tion for UGXT was glyoxylate detoxification. The glycine produced by the UGXT reaction can have a variety of metabolic fates in the cell. As for the other product of the UGXT reaction, oxalurate, a pathway has been described for the production of carbamoylphos-phate from this compound2, but the genes and proteins involved in the pathway have not been identified.

In the UGXT-catalyzed transamination, spontaneous con-version of the amino donor ((S)-ureidoglycine) can yield the acceptor molecule (glyoxylate) necessary to complete the reaction. A scheme of this new transamination sequence is proposed on the basis of biochemical and structural evidence and the known chemistry of the PLP cofactor (Scheme 1). The transamination of ureido glycine in vivo could also occur with already available keto acids that UGXT can use as amino group acceptors. However, during carbon starvation, or when purines are the only nutrient source, formation of glyoxylate from ureidoglycine should repre-sent the only way of carbon flux through the metabolic pathway. UGXT has considerably broadened the use of keto acid sub-strates with respect to its relatives, which are rather specialized on glyoxylate (see Fig. 4a). The use of different acceptors is consis-tent with a role of UGXT in nitrogen recovery, allowing forma-tion of different amino acids without energy costs in condition of nitrogen starvation and abundance of carbon sources. Hence, in the case of UGXT, the evolutionary pressure on the enzyme must have been two-fold: improving the ability to use different amino group acceptors for times when the recovery of nitrogen

YIYYYYYYYYYNNNNNNNNYYYWWWWFVSNNYYYYYYYYYFFWWWW

*

*

**

*

*

**

Plants

Agxt1_H.sapiens_1H0CAgxt1_M.musculus_3KGX

Agxt1_D.rerioXP_001630921_N.vectensis

2HUF_A.aegyptiNP_495885_C.elegans

1VJO_NostocNP_485047_N.muscorumYP_172644_S.elongatus

P9301_09401_P.marinusYP_291337_P.marinus

ZP_00833776_Y.intermediaCJA_0577_C.japonicus

Krad_2078_K.radiotolleransNP_421405_C.crescentusGOX1298_G.oxydans

YP_001488121_B.pumilusEF2994_E.faecalis

5114469SMa1495_S.melioti

DR1350_D.radioduransAgat_A.thaliana

XP_002279236_V.viniferaABQ81922_C.sativus

NP_001148339_Z.maysSgat_H.metylovorum

XP_001702107_C.reinhardtii2220311

3562082565365

XP_391069_G.zeaeXP_752314_A.fumigatusXP_758768_U.maydis

XP_001831648_C.cinereaAgx1p_S.cerevisiae_2BKW

YP_628537_M.xanthusTVN0571_T.volcanium

ZP_05569796_F.acidarmanusYP_254959_S.acidocaldarius

TneuDRAFT_0668_T.neutrophylus2Z9U_M.loti

NP_459426_S.typhimurium_1M32YP_002921917_K.pneumoniae

YP_856469_A.hydrophilaZP_06049730_V.cholerae

Pucg_B.subtilis

Metazoa Fungi Prokaryotes

AGXT12.6.1.51

UGXT

SGAT2.6.1.45

AEPT2.6.1.37

HHHHHHTTSSSRRRRRRRRSSSTTTTTTTTTTTTTTTTTTMVSSST

WWWWWWFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFYYYYYY

RRRRRRRRRRRLLLLLLLLRRRLLLLLLRRRGSSSRRRRRGGRRRR

7499 266

102

20 PAM

SHHHHHHHHHHQQQQQQQQHHHYYYYMHHHHHHHHHHFFFDDWWWW

39a

wt

Q39H

N266S

N266Y

b 2.0

1.5

1.0

0.5Abs

orba

nce

at 2

20 n

m

00 5 10 15

Time (min)

20 25 30

Figure 4 | Evolution of substrate specificity in UGXT proteins. (a) Phylogenetic tree of UGXT homologous proteins. Active site residues that differ between UGXT and AGXT proteins are indicated for each sequence of the tree. Sequences are labeled with GenBank or Microbesonline codes; known three-dimensional structures are indicated by asterisks. Enzymatic activities are indicated in accordance with the B6 database26. (b) Enzymatic activities of wild-type and mutated UGXT. Formation of oxalurate was monitored spectrophotometrically in the presence of AllC (1 μM), allantoate (0.3 mM), glyoxylate (0.3 mM) and 0.1 μM of wild-type UGXT or Q39H, N266S and N266Y mutants. The reaction was initiated by the addition of AllC (arrow).

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is a primary metabolic request and retaining a high efficiency toward glyoxylate, which is useful in harder times of nitrogen and carbon deprivation.

Given that spontaneous deamination of ureidoglycine would yield a stable compound (ureidoglycolate) during spontaneous decay of ureidoglycine into glyoxylate, the release of urea must precede the release of ammonia, with formation of a hydroxy-glycine inter mediate (Scheme 1). Kinetic data of ammonia release in the presence and in the absence of urease are consistent with this pathway (see Supplementary Fig. 5). Hydroxyglycine in bio-logical systems has been so far described at the C-terminal position of polypeptides as the product of peptidylglycine monooxygenase (EC 1.14.17.3). The peptidyl-hydroxyglycine product spontane-ously dismutates to glyoxylate and the corresponding desglycine peptide amide, a reaction catalyzed by peptidyl-amidoglycolate lyase (EC 4.3.2.5).

The spontaneous conversion of (S)-ureidoglycine to glyoxylate, which is quite rapid at neutral pH (~4 × 10−4 s−1), could be catalyzed in vivo. Genome comparisons suggest that some organisms, such as Enterococcus fecalis, are able to catalyze this reaction (see Fig. 1b), whereas many others, such as B. subtilis and Klebsiella pneumonie, do not possess candidate genes for ureidoglycine hydrolysis and may thus rely on spontaneous conversion of ureidoglycine into glyoxylate. The enzymatic activity on ureidoglycine of a K. pneumoniae UGXT homolog has been reported during the revision of this paper19.

Although UGXT seems to provide an effective solution for the use of oxidized purines, genes encoding this protein are only found in certain bacteria. A possible explanation, consistent with an origin of UGXT after the emergence of metazoa (see Fig. 4a), is a late appearance of UGXT-encoding genes and their association with particular ecological niches. For a long evolutionary period, the bioavailability of oxidized purines must have been limited, as purine bases are preferentially recycled through the salvage path-way or fully degraded to ammonia and glyoxylate. This situation changed dramatically with the appearance of animals that excrete oxidized purines to get rid of excess nitrogen. In many animals, including insects, birds, reptiles and mammals, part of the purine waste is transferred into the intestine, where it constitutes a poten-tial nutrient source for microorganisms20,21. Through the UGXT reaction, microorganisms can use oxidized purines as a source of carbon, nitrogen and energy, illustrating a perfect example of one species producing waste that another species can use as food. B. subtilis, along with other Firmicutes, is a member of the animal gut flora11, and spores of B. subtilis and related Bacilli are currently used for therapy of gastrointestinal disorders22. In humans, one-third of uric acid is excreted in the gastrointestinal tract23. High levels of uric acid in humans can cause gout and kidney disease. The presence of an efficient pathway for the use of oxidized purines in

microorganisms that can be administered to humans suggests that certain probiotics could also be tested for the ability to reduce uric acid levels and prevent gout.

METhoDSSynthesis of labeled compounds and NMR spectroscopy. Uniformly labeled [15N,13C]allantoate was obtained from [15N,13C]-(R)-adenosine (Spectra Stable Isotope) using seven enzymatic steps, as previously described5. Specifically labeled [2-13C]allantoate was obtained enzymatically from [8-13C]urate, which was synthesized by condensing 5,6-diaminouracil with [13C]urea (Sigma) according to a described protocol24. Labeled ureidoglycine was prepared in situ by adding 50–100 μg of recombinant allantoate amidohydrolase (AllC)5 to 0.6-ml solutions containing uniformly labeled allantoate or specifically labeled allantoate (4–11 mM). Reactions were conducted in 0.1 M potassium phosphate, 80% D2O, pH 8.0. Uniformly labeled [13C]glyoxylate was obtained enzymatically from [15N,13C] allantoate after 2 h incubation with AllC. Unlabeled authentic oxalurate for 13C spectroscopy was obtained from parabanate (Sigma) after 1 h incubation at 90 °C25. NMR spectra were collected as described in the Supplementary Methods. All spectra were consistent with literature values4,5 or with values obtained from chemical standards (Supplementary Fig. 2).

Bioinformatics. Search and comparison of genetic clusters containing allantoate amidohydrolase were conducted using the MicrobesOnline web server (http://microbesonline.org). UGXT sequences and sequences of related protein families were retrieved through homology searches from MicrobesOnline or from GenBank. Assignment of protein sequences to PLP-dependent enzymatic activities was made using the vitamin B6 database26. Sequence alignments were carried out with ClustalW27 and visualized with ESPript28. Phylogenetic analysis was performed using the neighbor-joining method29 implemented in ClustalW, and the resulting tree was visualized with FigTree (http://tree.bio.ed.ac.uk). Identification of structural similarity and pairwise structural alignment were performed with FATCAT30. PyMOL (http://pymol.sourceforge.net) was used to study and visualize protein models, and Coot31 was used to study electron density maps.

Recombinant expression and purification of PucG. The pucG gene was amplified by PCR from B. subtilis and cloned in the pET11b vector for recombinant expres-sion in E. coli, as described in the Supplementary Methods. The expression of PucG was induced at an optical density at 600 nm of 0.6 with 1 mM isopropyl-1-thio-β-D-galactopyranoside; after 4 h at 28 °C the cells were resuspended in 60 ml of sonication buffer (50 mM sodium phosphate, 0.3 M NaCl, 10% (v/v) glycerol, 1 μM pepstatin, 1 μM leupeptin, 100 μM PMSF, 40 μM PLP, 2 mg ml−1 lysozyme, pH 8) and incubated on ice for 30 min. The cells were lysed by four 15-s bursts of sonication. PucG was purified to homogeneity, as assessed by 15% SDS-PAGE analysis, by gel filtration (Sephadex-G100, Pharmacia) and by anion exchange chromato graphy (Q Sepharose, Pharmacia), with a final yield of approximately 6 mg l−1 of cell culture. The ε280 nm (extinction coefficient) of PucG was estimated to be 42,400 M−1 cm−1 on the basis of the amino acid sequence using the ProtParam pro-gram32 of the Expasy proteomics server (http://expasy.org/tools/protparam.html).

The Q39H, R74H, N266Y and N266S PucG variants were generated by site-directed mutagenesis as described in the Supplementary Methods.

Spectrophotometric assays. Reactions were monitored by measuring the increase of absorbance at 220 nm using a Varian Cary 1E spectrophotometer. At this wave-length, most α-keto acids possess an absorption coefficient approximately ten-fold higher than that of glyoxylate; a value of 4,780 M−1 cm−1 was used as the difference of the extinction coefficients of oxalurate and glyoxylate at 220 nm. The typical reaction mixture contained 0.1 μM PucG, 0.3 mM allantoate, 5 μM MnCl2,

(S)-Ureidoglycine

First partial reaction

Externalaldimine

Glyoxylate Oxalurate

Externalaldimine

Second partial reactionGlycine

Urea

Internalaldimine

Enzyme

Solution

O

NH

O

NH

H

O

OHH

O

O H

OH

Lys200 O

NH

+

HN+

+

–O–O

–O –O–O

–O

–O –O–O

–O

NH

O

H N+

3

2+

H N3

NH3

H O2

NH2+

3H NH

P+NH

OH

2NH

H O2

2NH

Lys200

O

NH

OH+HN

P

NH

OH

2NH

Lys200O

NH

O

2NH

2NH

P

O

O

NH

O

2NH

O

+NH

OH

2NH

Lys200

2NH

P

O

O H2H O 2NH

Lys200

+NH

OH

O

+HN

P

HH

Internalaldimine

+NH

OH

+HN

Lys200

P

O

+

3H N HH

Scheme 1 | UGXT reaction sequence.

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806 nature CHeMICaL BIOLOGY | vol 6 | NovEMBER 2010 | www.nature.com/naturechemicalbiology

article NATURE chEMicAL bioLoGy dOI: 10.1038/nCHeMBIO.445

0.3 mM glyoxylate or alternative amino group acceptors α-ketoglutarate, oxalo-acetate, hydroxypyruvate and pyruvate) in 1 ml of 20 mM potassium phosphate, pH 7.6. The reaction was initiated by the addition of AllC (0.4–1 μM) to obtain the unstable (S)-ureidoglycine substrate5.

To evaluate the PucG reaction in presence of amino group donors other than ureidoglycine, spectrophotometric assays were carried out in the presence of 0.26 μM PucG, 10 mM glyoxylate, 2 mM amino acid (Asp, Glu, Ser, Val, Ala) in 1 ml of 20 mM potassium phosphate (pH 8.5), and the increase of absorbance at 220 nm was recorded. The activity of AGXT1 on ureidoglycine was tested with the 220 nm assay described above. AGXT1 (0.11 μM) was added 10 min after the addition of AllC (0.65 μM) to a reaction mixture containing 1 mM allantoic acid, 5 μM MnCl2, 10 mM glyoxylate in 1 ml of 20 mM potassium phosphate (pH 8.5). Reversibility of the PucG reaction was tested spectrophotometrically using an excess of glycine (~50 mM), without observing consumption of oxalurate. This is consistent with the near-irreversibility of the AGXT-catalyzed transamination, which has an equilibrium constant >9,000 (ref. 18). Formation of glycine through the glycine oxidase assay and analysis of the spontaneous decay of ureidoglycine are described in the Supplementary Methods.

PucG crystallization, structure determination and modeling. Single crystals of PucG were obtained in 2–3 days at 20 °C by the vapor diffusion method after equilibrating a solution containing PucG (7 mg ml−1, in the presence of 15% (w/v) PEG monomethylether 550, 0.05 M NaCl, 0.05 M bicine, pH 9.0) against 30% (w/v) PEG monomethylether 550, 0.1 M NaCl, 0.1 M bicine, pH 9.0 (precipitant 1 of Structure Screen 2, Molecular Dimensions Ltd.). X-ray data collection and structure determination are described in the Supplementary Methods. Data collection and structure refinement statistics are summarized in Supplementary Table 2.

The model of (S)-ureidoglycine bound at the PucG active site was constructed manually by superimposing the carboxyl and amino groups of ureidoglycine to equivalent groups of enzyme-bound AGXT ligands7,15 (PDB: 1H0C and 2HUU). The conformation of the ureido side chain of ureidoglycine was modeled using the conformation of the ureido group of (S)-allantoin as a reference33 (PDB: 2Q37). The Coot program31 was used for manual model building, and the PRODRG2 server34 (http://davapc1.bioch.dundee.ac.uk/prodrg/) was used to add explicit hydrogens to the PucG structure.

Accession codes. The UGXT sequence has been deposited in GenBank with the accession code GQ303361. The coordinates of the UGXT structure have been deposited in the RCSB Protein Data Bank with the accession code 3ISL.

received 29 March 2010; accepted 26 august 2010; published online 19 September 2010

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acknowledgmentsWe thank L. Borghi, A. Nouvenne and F. Albertini for discussions, and the staff of beam-line XRD1 of Elettra, Trieste, for technical assistance during data collection.

author contributionsI.R. and R.C. performed experiments. L.C., R.B. and G.Z. performed the crystallographic studies. R.P. and A.P. designed experiments. R.P. conceived the study and wrote the paper with contributions from A.P. and G.Z. All authors analyzed data, discussed results and approved the final manuscript.

Competing financial interestsThe authors declare no competing financial interests.

additional informationSupplementary information is available online at http://www.nature.com/naturechemicalbiology/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to R.P.

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