The Structure and Function of a Microbial Allantoin Racemase Reveal

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Structure and function of a microbial allantoin racemase reveal the origin and conservation of a catalytic mechanism Laura Cendron, Ileana Ramazzina, Vincenzo Puggioni, Eleonora Maccacaro, Anastasia Liuzzi, Andrea Secchi, Giuseppe Zanotti, and Riccardo Percudani Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00881 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Biochemistry

Structure and function of a microbial allantoin racemase reveal the origin and conservation of a catalytic mechanism Laura Cendron1,4,a, Ileana Ramazzina2,5,a, Vincenzo Puggioni2, Eleonora Maccacaro2, Anastasia Liuzzi2, Andrea Secchi3, Giuseppe Zanotti1, Riccardo Percudani2*.

1

Department of Biomedical Sciences, University of Padova, Padova, Italy.

2

Department of Life Sciences, University of Parma, Parma, Italy.

3

Department of Chemistry, University of Parma, Parma, Italy.

4

Present address: Department of Biology, University of Padova, Padova, Italy.

5

Present address: Department of Biomedicine, Biotechnology and Translational Research, University of Parma,

Parma, Italy. a

Equal contribution authors

*Corresponding author: Riccardo Percudani, Department of Life Sciences, Parco Area delle Scienze 23/a, University of Parma, 43214 Parma, Italy. Email: [email protected] Tel: +39 0521 905140; FAX: +39 0521 905151

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Abstract The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in non-hominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted by glycine, yet the recombinant enzyme displayed racemization activity with similar efficiency (kcat/KM ≈ 5 x 104 M-1 s-1) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13C and 1H NMR, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by seven orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution, and conserved in spite of the substitution of catalytic residues.


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Introduction A single enantiomer of allantoin is produced in nature through uricolysis, a three-step purine degradation pathway whose last reaction is the enantioselective decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5ureidoimidazoline (OHCU) to give (S)-allantoin (1–4). With few exceptions (5), the enzymes for allantoin breakdown (allantoinase), though encoded by two different gene families, specifically catalyze hydrolysis of the S enantiomer (6,7). However, racemic allantoin is produced by the chemical oxidation of urate or its incomplete enzymatic conversion catalyzed by urate oxidase (8,9). In addition, if allantoin is not hydrolyzed, but rather eliminated in the environment, the R enantiomer is produced by spontaneous racemization, which occurs at a rate constant of ~2 x 10-5 at neutral pH (10). Remarkably, two separate mechanisms exist for allantoin racemization: formation of a bicyclic intermediate that can decompose to form either enantiomer of allantoin, and proton exchange at the chiral center with solvent. Both mechanisms contribute to spontaneous racemization, but the pathway through the bicyclic intermediate is about ten times faster than the other one (10). Allantoin can be utilized by various organisms as a nutrient source (11). Only a half of the racemic mixture can be readily utilized through stereospecific allantoinase, unless an enzyme is present that is able to catalyze the interconversion of allantoin enantiomers. Enzymes with allantoin racemase (EC 5.1.99.3) activity have a potential use in the determination of the output of urate oxidation. (S)-Allantoin can be quantified in serum by an enzymatic method based on the use of stereospecific allantoinase (12). The use of allantoin racemase would enable the quantification of both enantiomers and the distinction between enantioselective and non-enantioselective urate degradation. This can be useful for monitoring uricolytic activities in biological fluids after administration of uricolytic drugs in the enzymatic therapy of severe hyperuricemia caused by tumor lysis syndrome and Lesch-Nyhan disease (13,14). In early studies, isolation of the (R)-allantoin enantiomer through the use of stereospecific allantoinase (15), allowed testing several Pseudomonas species for growth on this substrate; some species were found to utilize this substrate as a nitrogen source due to the presence of a racemase enzyme (5). More recently, the Dcg1p gene of Saccharomyces cerevisiae and the hpxA gene of Klebsiella spp. were predicted to encode allantoin racemase (AllR) for their proximity with other genes involved in purine degradation and sequence similarity with other racemases (16,17). The demonstration of the AllR function was provided by the functional and structural characterization of the K. pneumoniae enzyme (18). The KpAllR protein is able to catalyze allantoin racemization with similar kcat and KM values for both enantiomers. The structure of the protein in complex with allantoin identifies two cysteine residues (C79 and C184) situated on opposite sides of the substrate chiral center in a position consistent with the two-base mechanism typical of the Asp/Glu racemase superfamily. Replacement of either of the two cysteines with serine leads to the loss of the enzymatic activity (18). The KpAllR mechanism is confirmed by quantum mechanics/molecular mechanics (QM/MM) calculations of the enzyme-catalyzed reaction, suggesting that allantoin stereoinversion proceeds through a stepwise mechanism involving a transient unprotonated intermediate (19). Here we present evidence, obtained through bioinformatics analysis, for the identification at the molecular level of the allantoin racemase enzyme (PfAllR) originally described in Pseudomonas species. This evidence ACS Paragon Plus Environment

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includes sequence similarity with the characterized KpAllR protein. Interestingly, however, the candidate PfAllR and orthologous proteins show the substitution of the first cysteine of the catalytic dyad with glycine. In spite of this non-conservative substitution, NMR analysis demonstrated that the PfAllR enzyme is able to catalyze proton exchange at the allantoin chiral center, while not affecting the velocity of the formation of bicyclic intermediate –an alternative faster mechanism of allantoin racemization. The atomic structure of the PfAllR enzyme provided a rationale for the enzyme activity, identifying a glutamate residue that can functionally replace the substituted cysteine in catalysis. The evolution of allantoin racemase within the Asp/Glu racemase superfamily illustrates a clear example of the importance of the enzyme ancestry for the choice and conservation of a particular catalytic mechanism.

Experimental procedures Bioinformatics. The analysis of genotype/phenotype correlation was conducted with the OrtholugeDB web server (www.pathogenomics.sfu.ca/ortholugedb) (20) by selecting completely sequenced organisms that were reported to possess or not possess the AllR activity (5). The complete set of orthologous genes was “filtered” according to the reported phenotype to obtain a list of orthologs present in AllR+ and absent in AllR- organisms. The presence of the identified genes in three genomes not included in Ortholugedb (P. tolaasii, P. alcaligenes, and P. pseudoalcaligenes) was determined through reciprocal best hits blast searches. Comparative analysis of the gene context was carried out with the MicrobesOnline web server (http://www.microbesonline.org) (21). Sequence alignments and phylogeneic trees (based on neighbor-joining of corrected genetic distances) were generated using Clustalx (22). Sequence logos for different groups of the Asp/Glu superfamiliy were generated using the WebLogo server (http://weblogo.berkeley.edu) (23). Protein expression and purification. DNA encoding for the candidate AllR was amplified from Pseudomonas fluorescens type strain (DSM No: 50090). Genomic DNA was prepared using described procedures (24) and the region encoding putative AllR protein was PCR-amplified using Go Taq DNA polymerase (Promega) and two sequence specific primers (supplementary Table S2). The amplification product was inserted into the pGEM vector (Promega) to generate the intermediate vector pGEM-AllR. The restriction fragment obtained from NdeI/BamHI digestion was then ligated into the expression vector pET11b (Novagen). The resulting plasmid was verified by sequencing and electroporated into E. coli BL21 codon plus cells (DE3). The expression of AllR was induced at an optical density at 600 nm of 0.6 with 1 mM IPTG; after 5 h at 28 °C the cells were resuspended in 180 mL of lysis buffer (50 mM sodium phosphate, 0.3 MNaCl, 1mM β-mercaptoethanol, 10% glycerol, 1 µM pepstatin, 1 µM leupeptin, 100 µM phenylmethylsulfonyl fluoride, pH 8) and lysed by sonication. The supernatant obtained after centrifugation of the crude extract was concentrated by ultrafiltration in an Amicon cell (YM-10 membrane, Millipore). AllR was purified to homogeneity, as assessed by 16% SDS-PAGE analysis, by anion exchange (Q Sepharose, Pharmacia) and gel filtration (AcA54, Pharmacia) chromatography, with a final yield of approximately ~6 mg L−1 of cell culture. The anion exchange column (10 x 1.5 cm) was equilibrated in 0.1 M KP, pH 7.6 and eluted with a linear gradient of NaCl (0-0.5 M in 60 mL of column buffer). Fractions corresponding to ~0.3 M NaCl, were concentrated by ultrafiltration in an Amicon cell (YM-10 membrane, Millipore), applied on to the AcA54 column (120 x 1.5 cm) and eluted with 0.3 M NaCl in 0.1 M KP, pH 7.6. The


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Biochemistry

ε280 nm (extinction coefficient) of AllR was estimated to be 15930 M1 cm1, based on the amino acid sequence. The molecular weight of purified AllR was estimated by gel filtration on a Sephadex G-100 column (Pharmacia) (55 x 1.5 cm), using 0.1 M potassium phosphate (pH 7.6) as buffer for equilibration and elution. Crystallization and data collection. Initial crystallization screens were carried out at 20°C by vapor diffusion technique using an automated sitting-drop setup (Oryx-8, Douglas Instruments). The most promising conditions were reproduced and optimized by hanging-drop method. The best crystals grew in two conditions of the Structure screen I and II HT-96 kit (Molecular Dimension Ltd): SSI n.38 (0.1 M Tris pH 8.5, 8% w/v PEG 8000) and SSI n.23 (0.2 M CaCl2, 0.1M HEPES pH 7.5, 28% w/v PEG 400), corresponding to two different crystal forms which were grown. Cubic crystals (a=b=c=109.72 Å), grown in SSI n.38 condition, belong to the P213 space group and contain two monomers per asymmetric unit (VM=2.21 Å3 Da-1, solvent content 44%). Orthorhombic crystals (P212121, a=60.22Å, b=142.32Å, c=146.02Å), grown in SSI n.23 condition, contain an entire hexamer in the asymmetric unit, corresponding to a VM of 2.08 Å3 Da-1 and a solvent content of about 41%. Data for the cubic and orthorhombic crystal forms were measured, respectively, at the ID23-1 and ID14-1 beamlines of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Both crystal forms were cryoprotected by soaking for a few seconds in the corresponding crystallization condition supplemented with 20% glycerol, prior to flash freezing and storing in liquid nitrogen. The best crystals diffracted to a maximum resolution of 2.10 Å and 2.15 Å for the orthorhombic and the cubic crystal forms, respectively. Structure determination and refinement. All datasets were indexed and integrated with Mosflm software (25) and merged and scaled with Scala (26), contained in the CCP4 crystallographic package (27). Both structures were solved by molecular replacement using the Phaser software (28). The model was displayed and manually adjusted with graphic Coot software (29). Refinement was carried on using Refmac (30) and Phenix (31) packages. The two structure were refined to a final R factor of 0.205 (Rfree 0.264) and 0.188 (Rfree 0.233) for the cubic and orthorhombic crystal forms, respectively. Geometrical parameters of the models, checked with Procheck (32), are as expected or better for this resolution. Data collection and refinement statistics are summarized in Table 3. Synthesis of allantoin enantiomers. (S)-Allantoin was synthesized enzymatically from urate. A typical reaction mixture (10 mL, 100 mM KP, pH 7.6) contained 5 mg/mL urate, 10 mg of Uox from Candida (Sigma), 2.5 mg of catalase, recombinant Urah (0.1 mg) and Urad (1 mg) form zebrafish (2,33). The mix was incubated under stirring at room temperature; the complete conversion of uric acid to allantoin was monitored by collecting spectra between 200 and 340 nm. (R)-allantoin was synthesized enzymatically starting from racemic allantoin. A typical reaction mixture (10 mL, 100 mM KP, pH 7.6) contained 10 mg/mL of allantoin and recombinant allantoinase (0.2 mg) from P. fluorescens (6). Proteins were removed from the solution by Amicon (10 kDa cutoff). The prepared enantiomers were utilized immediately in enzymatic assays or briefly stored at -80 °C for the determination of the reaction velocity at different substrate concentrations. The (R)-allantoin preparation contained allantoic acid; however, the AllR reaction was reported not to be inhibited by this compound (18). Site-directed mutagenesis. The site-directed mutants E78Q and E78D were obtained by site-directed mutagenesis using a mix (50 µl) containing 50 ng of the PfAllR expression vector, 2.5 U of PFU turbo (Stratagene, La Jolla, CA, USA), 0.3 mM dNtp, 2% DMSO and 126 ng of each mutagenic primers (supplementary Table S2). Reactions were performed as follows: initial denaturation at 95 °C (30 s) followed by 16 cycles of 95 °C (30 s), ACS Paragon Plus Environment

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55 °C (60 s) and 68 °C (7 min), followed by 5 min at 68 °C. For each mutation, the product of reaction was treated with DpnI (New England Biolabs) to digest the parental DNA template. The products of each digestion were used to transform E. coli XL1 Blue cells. Single clones were sequenced to confirm the presence of the desired mutation. CD spectroscopy. Circular dichroism measurements were carried out at 25 °C in a 10-mm light-path length cuvette with a Jasco J715 spectropolarimeter. AllR activity was determined using (S)-allantoin or (R)-allantoin as substrates in the presence of AllR (9 µg) in 1 mL of 0.1 M potassium phosphate (pH 7.6). CD spectra were recorded in the 200-300 nm range for 10 min, every minute. To evaluate the kinetic parameters of wild type and mutants enzymes, ellipticity of allantoin enantiomers (6) was monitored at a fixed wavelength of 220 nm (molar ellipticity = 2.1 x 103 degrees M-1 m-1) for concentrations ≤ 0.6 mM and of 240 nm (molar ellipticity = 0.2 x 103 degrees M-1 m-1) for higher concentrations. The reactions were initiated by the addition of AllR (1.8 µg) to 1 mL of 0.1 M potassium phosphate (pH 7.6). The reaction rate was obtained for different substrate concentrations by linear fitting of data points collected over the first 15 s of the reaction. C and 1H NMR assays. Labeled [2-13C] (S)-allantoin, used for 13C NMR assays, was obtained enzymatically from [8-13C]urate, which was synthesized by condensing 5,6-diaminouracil with [13C]urea (Sigma) according to a described protocol (8). Labeled urate was converted to [2-13C] (S)-allanotin in the presence of 20 units of urate 13

oxidase from Candida utilis, 28 units of catalase from Corynebacterium glutamicum (Fluka), 12 µg of zebrafish HIU hydrolase and 156 µg of zebrafish OHCU decarboxylase. The conversion was performed in 1.1 mL of 0.1 M potassium phosphate (20% D2O, pH 7.4) and monitored spectrophotometrically. After the completion of the reaction, the solution was ultrafiltrated to remove proteins and then transferred into a 5-mm NMR tube. The time course of isotopic exchange of [2-13C] (S)-allantoin was monitored in the absence and in the presence of 9 µg of recombinant AllR. The 13C NMR spectra were proton decoupled and were collected at 25 °C with a VARIAN Inova 600 instrument. 1H NMR assays were performed in 0.55 mL of 0.1 M potassium phosphate (98% D2O, pD 7.6), containing 20 mM of (R,S) allantoin, in the absence and in the presence of 5.2 µg of recombinant AllR. The time courses of proton-deuterium exchange were collected at 25 °C with a Bruker AVANCE 300 instrument.

Results Bioinformatics evidence for the identification of Pseudomonas allantoin racemase. Allantoin racemase activity was first described in Pseudomonas species (5), but the genes/proteins responsible for the activity in these organisms have not been identified at the molecular level. By homology searches with the sequence of a confirmed AllR protein of K. pneumoniae (18), we found several proteins with significant similarity in Pseudomonas spp. The best hit in P. fluorescens, was a protein sharing 40% sequence identity with KpAllR (Figure 1A). Comparative analysis of the gene context provided additional evidence for the functional identification (Figure 1B). In Pseudomonas, the gene is encoded in the same operon with a gene similar to the DAL4 allantoin transporter of Saccharomyces cerevisiae (34); in other organisms, the gene is found near genes similar to characterized allantoinase and allantoicase.


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Biochemistry

In the original study, the AllR activity was meticulously assayed in 17 Pseudomonas isolates and found to be scattered across species (5). Several complete genomes are available of the species/strains tested for the AllR activity (supplementary Table S1), allowing to search for genotype/phenotype correlations. A whole genome analysis of orthologous genes (20) identified the candidate PfAllR among the four genes (ADE88156, PFLU0825, PFLU2141, and PFLU2444) present in AllR+ species and absent in AllR- species (Figure 1C). Evidence from gene context and genotype/phenotype correlation would have allowed the functional identification of the gene even in the absence of a characterized homolog. Paradoxically, the very examination of the homology evidence calls this identification into question. The Pseudomonas protein lacks a cysteine residue (C79 in the KpAllR sequence) that is essential for the two-base catalytic mechanism of KpAllR. The residue is substituted by glycine in the candidate PfAllR sequence (see Figure 1A), while the more conservative Cys79Ser substitution was found to completely abolish the enzyme activity (18). The Cys79Gly substitution is observed in an ungapped block of the alignment in multiple homologous sequences, thus ruling out the possibility of sequencing or misalignment errors.

Recombinant expression and activity of PfAllR In the presence of contrasting evidence from bioinformatics analysis, we decided to test the activity of the isolated protein. The candidate gene was cloned from Pseudomonas fluorescens type strain in Escherichia coli, and the corresponding untagged protein was overproduced and purified to apparent homogeneity by ionexchange and gel-filtration chromatography. Size of the protein as determined by gel filtration (~55 kDa) is consistent with a dimeric quaternary structure in solution. The purified protein, assayed through CD spectroscopy for activity on (R)- and (S)-allantoin substrates, was found to be able to catalyze the interconversion of the two enantiomers (Figure 2A), with kcat≈130 s-1 and KM =2.5 mM for S → R conversion and kcat≈60 s-1 and KM =1.1 mM for R → S conversion (Figure 2B and Table 1). Differences in the kinetic parameters are compensatory, so that the catalytic efficiency is about the same for the two enantiomers (kcat/KM ≈ 5 x 104 M-1 s-1) as reflected by the similar reaction progression curves (Figure 2C). Compared to the uncatalyzed reaction, the rate of allantoin racemization is enhanced by six orders of magnitude by the enzyme (Table 2).

Crystal structure of PfAllR The crystal structure of P. fluorescens allantoin racemase has been determined in two different crystal forms (cubic and orthorhombic) at 2.10 and 2.15 Å resolution, respectively (Table 3). The overall folding of the monomer (Figure 3A) is similar to that of other proteins of the Asp/Glu racemase superfamily: two domains, each containing a four-stranded parallel β-sheet sandwiched between two pairs of α-helices. The highest structural similarity is with the AllR enzyme from K. pneumoniae (18). The r.m.s.d. between equivalent Cα atoms of one monomer of our structures compared to one of the K. pneumoniae (PDB ID 3QVJ) is 1.30 Å and 1.29 Å for the cubic and the orthorhombic forms, respectively.


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The monomeric structure of PfAllR in the two crystal forms is virtually the same, the r.m.s.d. between the Cα atoms of the dimer of the cubic form and one of the three dimers present in the orthorhombic form being 0.41 Å. In the orthorhombic form, the electron density is clearly visible from residue 1 (Met) to 236, except for residues from 144 to 153 in chain A and G (chains of the three dimers are labeled A-B, C-D, F-G). In the same two chains, some disorder is also present in the region 180-190. The latter area includes Cys 180, one of the key residues in the active site. In the cubic form, area 180-190 is well defined, including Cys 180. There is extra electron density in correspondence of the sulfur atom of this latter residue, suggesting that the cysteine sulfur is oxidized (possibly to sulfinic acid). The region 144-151 is characterized by poor electron density and high Bfactors. This flexible region could have a role in controlling the access of the substrate at the active site. The P. fluorescences allantoin racemase presents the same quaternary structure in the crystal state of the enzyme from K. pneumoniae: six monomers arranged as a trimer of dimers around a three-fold axis (Figure 3B,C). The latter is a crystallographic axis in the case of the cubic crystal form, and a non-crystallographic symmetry axis in the case of the orthorhombic form. An analysis performed with the PISA server indicates that the enzyme has a hexameric assembly in the crystal. The area buried in the complex formation is 24,150 Å2 over a total accessible surface of 41,960 Å2. This is in perfect agreement with the quaternary assembly observed in K. pneumoniae allantoin racemase However, a striking difference between the two proteins is observed at the active site, identified by superimposing the protein backbone of PfAllR to the KpAllR structure with a bound allantoin molecule (Figure 3D). With respect to the equivalent Asp 82 residue of KpAllR, the Glu 78 side chain of PfAllR points in the opposite direction, towards the substrate. The carboxylic group of Glu 78 nearly occupies the space taken by the thiol group of Cys 79 in KpAllR, a residue substituted by Gly in the P. fluorescens protein (see Figure 1A). The other residues that line the active site cavity, including the other catalytic cysteine, are conserved, both in terms of nature and conformation. The substitution of a carboxylic group for a thiol group at the protein active site could influence the mechanism of allantoin racemization in PfAllR.

Mechanisms of allantoin racemization. The atomic structure of PfAllR suggested that a glutamate residue (E78) could functionally replace the substituted cysteine in catalyzing proton exchange at the allantoin chiral center. Direct evidence of this mechanism of catalysis can be provided by NMR analysis. Allantoin can undergo racemization by two mechanisms that can be separately monitored by 13C and 1H NMR: formation and hydrolysis of a bicyclic symmetrical intermediate and proton exchange with the solvent (Figure 4A and supplementary Figs. S1-2). The first mechanism, which involves the scrambling between C2 and C7 atoms (respectively, 159.4 and 159.7 ppm at pH 7.4), was monitored by following the 13C signals of (S)-allantoin labeled at C2; the second mechanism was followed by monitoring the exchange of the C5 proton (5.27 ppm at pH 7.6) with solvent in deuterated water (Figure 4B). The velocity of racemization through the bicyclic intermediate was found to be independent from the presence of the enzyme (Figure 4C). According with previous results (10), the velocity of proton exchange was slower than the C2-C7 scrambling, but this velocity was increased by seven orders of magnitude by the enzyme (Figure 4D and Table 2).


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Biochemistry

To gain evidence of the involvement of E78 in the proton exchange mechanism catalyzed by PfAllR, this residue was replaced by site-directed mutagenesis with Asp (E78D) or Gln (E78Q). The mutant proteins were produced and purified using the same procedures as the wild type and assayed in the racemization reaction. The mutation with a shorter carboxylic side chain (E78D) resulted in a diminished catalytic activity (Figure 4E) that affected kcat and KM for both the enantiomers (see Figure 2A and Table 1). The mutation with the amide group (E78Q) resulted in the loss of catalytic activity (Figure 4E). These results are consistent with the conservation of the E78 residue in proteins belonging to the same orthologous group as PfAllR: a glutamate residue is present at equivalent positions except for an aspartate in a few cases. Based on the above evidence and the PfAllR structure, we conclude that the mechanism of racemization of PfAllR (Figure 4F) involves Glu 78 acting as a base for the abstraction of the C5 proton of (S)-allantoin and Cys 180 transferring a proton on the opposite side of the allantoin ring. In the stereoinversion of (R)-allantoin, proton abstraction is performed by Cys 180 and re-protonation by Glu 78. The charge of the oxyanion in the deprotonated intermediate (18,19), could by stabilized through H-bond interactions formed by the backbone NH of Gly 181 and Tyr 76 that in the PfAllR structure are in close proximity to the carbonyl oxygen on C5 of the docked substrate (see Figure 3D).

Origin and conservation of the catalytic mechanism of allantoin racemase. Experimental evidence indicates that AllR enzymes have been selected during evolution for the catalysis of allantoin racemization through a mechanism that is kinetically less favorable than an alternative one; PfAllR has conserved this mechanism in spite of the substitution of a catalytic residue. To gain insights into the evolutionary origin of the catalytic mechanism of AllR, we examined its phylogenetic relationships and distribution within the Asp/Glu racemase superfamily (Figure 5). For this analysis, we identified, through Hidden Markov Models (HMM) searches, 2702 Asp/Glu racemase members in a set of 1700 reference complete genomes. The protein phylogenetic tree shows four main groups; a tentative function can be assigned to groups or subgroups based on the presence of characterized proteins. The most numerous group (GLU) is represented by proteins assigned to glutamate racemase by similarity with the structural and functional characterized enzymes from Aquifex pyrophilus (PDB ID: 1B74), Helicobacter pylori (PDB ID: 2JFY), Escherichia coli (PDB ID: 2JFN), and Bacillus anthracis (PDB ID: 2DWU) (35–37). A second group (ASP) phylogenetically close to Glu racemase, is represented by aspartate racemase and includes the characterized enzyme from the archaeon Pyrococcus horikoshii (PDB ID: 1JFL) (38). Both glutamate and aspartate racemase exhibit strict conservation of the two catalytic cysteines (Figure 5). By contrast, in numerous sequences of a distinct subgroup allied to aspartate racemase, the first cysteine is often substituted by threonine or alanine. A member of this subgroup, named EcL-DER (PDB ID: 5ELL), has been recently reported to exhibit racemase activity with the L-enantiomers of Glu and Asp (39). A third group (MAL) includes characterized maleate isomerase from Nocardia farcinica (PDB ID: 2XEC) and Pseudomonas putida (PDB ID: 4FQ7) (40,41), and malonate decarboxylase from Bordetella brochiseptica (PDB ID: 3DG9) (42). The first cysteine of the catalytic dyad is typically conserved in the maleate


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isomerase subgroup but not in the malonate decarboxylase subgroup. Finally, a fourth group (HYD) includes sequences characterized as hydantoin racemase (AAQ93382, BD181026) (43,44) and allantoin racemase (KpAllR and PfAllR). The two enzymes catalyzing the racemization of allantoin (5-ureido hydantoin) form two distinct subgroups that are intermixed by sequences characterized as hydantoin racemase. A common characteristic of hydantoin and allantoin racemase is the presence of an acidic residue at position +3 with respect to the position occupied by N-terminal cysteine of the dyad; in the PfAllR subgroup, this residue is glutamate, while the cysteine is substituted by a conserved glycine (Figure 5). By constructing specific HMM profiles, we observed the occurrence of the four main phylogenetic groups of the superfamily (GLU, ASP, MAL, and HYD) in 143 completely sequenced organisms classified in the Tree of Life (45). The gene distribution (supplementary Figure S3) shows that the Asp/Glu superfamily is mostly represented by bacterial sequences. Among eukaryotes, genes belonging to the ASP group are found in plants, and genes belonging to the HYD group are found in fungi (including the putative allantoin racemase Dcg1p of Saccharomyces cerevisiae), while members of the superfamily are absent in animals. Indeed, in previous work, the presence of allantoin racemase sequences in the genomes of nematodes led to the identification of a contaminant bacterial genome (46). Genes belonging to the ASP, MAL, and HYD groups are found in some archeae. However, in the phylogenetic tree, these archaeal sequences cluster with bacterial sequences, suggesting that they could derive from lateral gene transfer. Overall, the data support an origin of the Asp/Glu superfamily in the common ancestor of all bacteria, with the GLU group as the founding member of the superfamily. This is consistent with the presence of orthologous genes of the GLU group in the vast majority of bacteria, and with the fundamental role of glutamate racemase in providing the D-glutamate building block of peptidoglycan. This defining feature of the bacterial cell is thought to be already present in the bacteria last common ancestor (47). A peptidoglycan cell wall has been lost by several bacterial species such as Tenericutes, Chlamydia, and Wolbachia. These species have also lost GLU racemase genes (supplementary Figure S3).

Discussion The observation of a substituted catalytic residue in protein sequence alignment is generally considered to be evidence that the protein has lost or modified the enzymatic activity. Before reaching this conclusion, the alignment is manually inspected to rule out problems arising from misalignment of gapped regions. In the case described here, the non conservative Cys/Gly substitution occurs in an ungapped block (see Figure 1A) and can be confidently considered as an evolutionary substitution. Consistent with data on the enzymatic activity, the atomic structure of the protein revealed a downstream residue (E78) that can functionally replace the missing catalytic residue in the two-base mechanism (see Figure 3D). These results emphasize the importance of the availability of structural information for protein functional annotation (48). Changes of the identity and position of catalytic residues during divergent evolution has been observed in "mechanistically diverse superfamilies” (49), such as enolase or Nudix hydrolase. These examples, however, concern distantly related proteins catalyzing different reactions. It is uncommon to observe changes in the ACS Paragon Plus Environment

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Biochemistry

identity and position of catalytic residues in similar proteins catalyzing the same reaction. It is not clear what selective advantage has driven the substitution of the catalytic cysteine in allantoin racemase. Certainly, the replacement of one of the two cysteines with glutamate creates a more asymmetric active site that could allow different reactivity towards the two enantiomers. The kinetic parameters for the two enantiomers appear more diverse for the enzyme with the carboxyl thiol dyad (PfAllR) with respect to the enzyme with the thiol dyad 4

-1 -1

(KpAllR), but the catalytic efficiency for the two enantiomers is similar in PfAllR (5 x 10 M s ) and inferior to that reported for KpAllR (6 x 105 M-1 s-1) (4). The phylogeny and distribution of Asp/Glu racemase superfamily in living organisms suggest that the original function of these proteins could be the synthesis of the D-glutamate component of the bacterial cell wall. At variance with an early origin of glutamate racemase, the enzymatic racemization of allantoin probably appeared late in evolution. For a long evolutionary time, the bioavailability of this intermediate of purine catabolism must have been limited, as in microorganisms purines are either recycled or completely degraded for carbon and nitrogen recovery (11). Allantoin, however, gained a special role in plants and animals as the end product of purine catabolism. Some plants (e.g. tropical legumes) use allantoin for nitrogen storage and transport, and some animals (e.g. non-hominoid mammals) use allantoin for the elimination of purine nitrogen. The divergent evolution of allantoin racemase from an Asp/Glu racemase progenitor could have allowed certain organisms (particularly bacteria and fungi) to adapt to the increased availability of this compound in the environment. The enzymatic racemization of allantoin illustrates a clear example of evolutionary choice among alternative mechanisms of catalysis (see Figure 4A). Interestingly, the mechanism selected for catalysis (proton abstraction) is kinetically less favorable than the formation of the symmetric intermediate –a reaction unexploited by the enzyme. It should be noted that in the initial phase of evolution, the presence of a mechanism of spontaneous racemization that is ten times faster the one catalyzed by the enzyme weakened the kinetic advantage of the proto-enzyme. A proto-AllR enzyme would not have provided selective advantage unless it had been able to increase the proton exchange rate by one order of magnitude. According to the evolutionary history of Asp/Glu racemase, proton abstraction at the chiral center and stabilization of a negatively charged enedienolate intermediate (35,50) could be considered the archetypal catalytic mechanism of the superfamily (see Figure 5). This mechanism is found in glutamate racemase, aspartate racemase, and malonate decarboxylase. Variation to the common theme are found in maleate isomerase, in which the proposed intermediate is a succinyl-Cys covalent enzyme-substrate complex (40,41), and in allantoin racemase, in which the proposed intermediate is an enolate. It should be remarked that the stabilization of the negative charge of the bicyclic intermediate could in principle allow an enzyme to catalyze racemization through the alternative reaction mechanism (see Figure 4A). However, QM/MM calculations of the KpAllR reaction suggest that the allantoin substrate binds the enzyme with an extended trans-ureido group (19), a conformation that is unfavorable for the nucleophilic attack of ureic nitrogen on the C4 carbonyl group and the formation of the bicyclic intermediate. The origin and evolution of enzyme catalysis is a problem of significant interest (51–53). When a catalytic mechanism of an enzymatic reaction is studied, a relevant question is why the enzyme developed that particular catalytic strategy. In many cases, the answer is that a protein using that catalytic strategy already


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existed that could be adapted for a novel substrate. As shown by the case presented here, this answer implies that alternative, viable mechanisms of catalysis exist that have not been selected by evolution. Thus, while conservation of structural/mechanistic aspects of catalysis appears to dominate the evolution of enzyme families, the choice of a particular catalytic mechanism for an enzyme can easily depend on its ancestry. The fact that novel functions are created by modification of old functions rather than by de novo invention, is a wellestablished notion in biology as epitomized by Jacob’s statement “Nature is a tinkerer, and not an inventor” (54). Although it is generally assumed that this notion also applies to the evolution of catalytic mechanisms, it is not easy to find examples in which the choice between alternative reaction mechanisms can be readily explained by the ancestry of the protein responsible for the catalysis. Allantoin racemase provides circumstantial evidence that the evolutionary origin of an enzyme can be the decisive factor in the choice of a particular catalytic mechanism.


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Acknowledgements We thank Claudia Folli for discussion and help with the experiments.

Funding Information This work was supported by the Telethon Grant GGP13149 to RP.

Database The sequence of Pseudomonas fluorescens AllR has been submitted to GenBank under the accession number ADE88156. Atomic coordinates and structure factors have been deposited at the Protein Data Bank for immediate release with ID codes 5FLD and 5LG5 for the cubic and orthorhombic crystal forms, respectively.

Supporting Information Table S1. Genome accessions of the Pseudomonas species previously tested for AllR activity. Table S2. Primers for PfAllR amplification and mutagenesis. Figure S1. 13C NMR data for the enzyme-catalyzed and uncatalyzed C2-C7 isotopic exchange of allantoin. Figure S2. 1H NMR data for the enzyme-catalyzed and uncatalyzed proton-deuterium exchange of allantoin. Figure S3. Organism distribution of the different groups of the Asp/Glu racemase superfamily.


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Table 1. Kinetic constants for wild type and mutant PfAllR.

PfAllR (S)-Allantoin -1 a

kcat (s )

-1 a

KM (mM )

-1 -1

kcat / KM (M s ) a

Glu78Asp

(R)-Allantoin

(S)-Allantoin

(R)-Allantoin

129±14

57±6

n.d.

43±7

2.5±0.6

1.1±0.3

n.d.

2.3±0.6

(5.2±1.8) x 10

4

(5.2±1.9) x 10

4

(1.6±1.1) x 10

4

(1.9±0.8) x 10

4

Kinetic parameters were determined by non-linear fitting of the data reported in Figure 2.

Table 2. Enhancement of macroscopic and microscopic reaction rate constants of allantoin racemization by PfAllR. Loss of optical a activity -1

kcat (s ) -1

kuncat (s ) Rate enhancement

129

Proton b exchange

C2-C7 isotopic c exchange -5

165

6.2 x 10 2 x 10

-5

6

≤7.4 x 10

1.1 x 10 1 x 10

-5

7

a

7.4 x 10

-5

≤1

Loss of CD signal measured at pH 7.6 in 100 mM KP in the presence and absence of the enzyme; kcat is from Michaelis-Menten kinetics. b Proton exchange with deuterium measured at pH 7.6 in 100 mM KP, 98% D2O in the presence and absence of the enzyme; kcat was calculated from the initial velocity obtained with 20 mM allantoin and 5.2 µg of PfAllR. c Isotopic exchange measured at pH 7.4 in 100 mM KP in the presence and in the absence of the enzyme.


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Table 3. Data collection and refinement statistics. Data set

Orthorhombic

Cubic

Wavelength

0.97372

1.0000

Space group

P212121

P213

a=60.22, b=142.32, c=146.02,

a=b=c 109.72,

6

2

39.78-2.10

77.58-2.15

(2.21-2.10)

(2.27-2.15)

0.068 (0.329)

0.080 (0.212)

11.4 (3.3)

19.5 (12.1)

Completeness (%)

99.4 (96.9)

97.4 (86.1)

3.8 (3.7)

9.4 (9.2)

73475

23619

0.188/0.233

0.205/0.264

Protein

10557

3505

Water

877

288

0.008

0.016

1.2

1.9

Favoured

94.9

92.2

Allowed

4.4

7.3

Generously allowed

0.6

0.5

0

0

0.2

0.0

Cell parameters (Å), number of monomers in a.u. Resolution (Å)

Rsym or Rmerge

Redundancy

Refinement No. reflections Rwork / Rfree No. atoms

R.m.s. deviations Bond lengths (Å) Bond angles () Ramachandran plot (%)

Outliers Overall G-factor


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Biochemistry

Figure legends Figure 1 Bioinformatics evidence for the identification of P. fluorescens AllR. (A) Alignment of K. pneumoniae AllR (3QVJ) with the homologous P. flourescens protein (ADE88156). Known residues involved into the two-base catalysis are indicated by blue arrows; functionally equivalent residues identified in the PfAllR structure (see Figure 3D) are indicated by red arrows. (B) Examples of genetic associations of the putative PfAllR and similar genes (white boxes) with genes involved in purine catabolism (colored boxes). (C) Genotype/phenotype correlation between the presence of PfAllR orthologs in complete genomes and the AllR phenotype. The tree is in accordance with the NCBI taxonomy. Figure 2. Racemase activity of P. fluorescens AllR. (A) Time course of S (red lines) and R (green lines) allantoin CD spectra in the presence of PfAllR (9 µg); spectra were taken every 60 s with 0.23 mM allantoin. (B) Kinetic □ ) allantoin in the analysis. Initial velocities were determined with different concentrations of S (○) or R (□ presence of 1.8 µg of wt PfAllR (left panel) or E78D mutant (right panel); data were fitted with the MichaelisMenten equation. The solid red line in the left panel is a tentative fitting yielding a KM higher than the maximal concentration used in the assay. The initial slope of the hyperbola (dashed line) was used to estimate kcat /KM. (C) Catalyzed and uncatalyzed reaction courses; dashed lines represent the loss of CD signal in the absence of PfAllR (kuncat=6.2 x 10-5 s-1), circles are experimental data points in the presence of PfAllR, red and green lines are kinetic curves (55) calculated as a function of time, initial substrate concentration, Vmax (3.5 x 10-7 mM x kcat ), and KM. Figure 3. Crystal Structure of PfAllR. (A) Monomer in cartoon representation. (B) Dimeric organization in the cubic crystal form; the hexamer is generated by the crystallographic 3-fold axis. (C) Hexameric organization in the orthorhombic crystal form. (D) Comparison of the active site of PfAllR in the cubic crystal form (left, cyan carbon atoms) and KpAllR (right, orange carbon atoms; PDB ID: 3QVJ). Enolic allantoin (gray carbon atoms) was docked by superimposing the backbone chain of the two protein structures to the protein-substrate complex of KpAllR double mutant (PDB ID: 3QVK). Hydrogen bonded contacts are indicated by gray dashed lines. Distances of the putative catalytic dyads to the allantoin chiral center are reported in angstrom. Figure 4. Uncatalyzed and catalyzed allantoin racemization mechanisms. (A) Alternative mechanism of allantoin racemization. The labeled carbon atom in the (S)-allantoin molecule is indicated by an asterisk. (B) Examples of the time-dependent isotopic exchange between C2 and C7 (upper panel) and H-C5 protondeuterium exchange (lower panel) as monitored by 13C and 1H NMR. (C) Kinetics of isotopic exchange between C2 and C7 of allantoin at pH 7.4 in the absence (empty circles) and in the presence (filled circles) of PfAllR. (D) Kinetics of C5 proton exchange in 98% D2O, pH 7.6 in the absence (right panel) and in the presence (left panel) of PfAllR. (E) Comparison of the activity towards the S and R enantiomers of allantoin (0.2 mM) for the wt and Glu 78 mutants (1.8 µg). (F) Proposed mechanism of allantoin racemization catalyzed by PfAllR. Figure 5. Evolution of allantoin racemase within the Asp/Glu racemase superfamily. Unrooted neighborjoining tree of the Asp/Glu racemase superfamily. Experimentally characterized proteins are indicated in bold with PDB or Genbank accessions; other sequences are indicated with Genbank accessions followed by the taxid. Sequences from eukaryotic organisms (Plants, Fungi) are labeled. Tree branches assigned to functional groups based on the phylogenetic analysis are shown in different colors next to sequence logos representing residue ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

conservation around the cysteine dyad (-3,+3). Reaction intermediates previously proposed based on structural evidence are shown in colored boxes.


N H

Page 23 of 28

H N

O NH2

H

N H

Biochemistry

H N

O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N H

O

H

N H

N H H N

-O

NH2

kuncat≈10-4 NO H

O

HN

H N

H N

ONH2 N H

O

N H

O

H N

H OH

O (S)-allantoin Glu78

N H

O

H

N H

H N

O-

S-N H

OH Glu78

O

OO

NH2

N H

N O H H

O

H H N N

-O

NH NH22

Cys180

H

N H

O

(R)-allantoin Cys180

OO

N N H H

N N H H OH Glu78

SH O

Cys180

O

H N

NH2

H N

O NH2

kuncatTyr76 ≈10-5 Gly181 NH 2 kNH cat≈10

OO

O

O

(R)-allantoin

O

SH

N H

O

H N

NO H

H

N H

O

NH2

NH2

O

(S)-allantoin

O

O

NH2

H N O

O

N N O N N Graphical abstract. Evolutionary choice between O H H H alternative H Tyr76 H H Gly181 reaction mechanisms in allantoin racemase OH NH SOSH

Glu78

O

NH

H N

OCys180

NH2

Glu78

O

O O

N H OH Glu78

O

N H SH Cys180


Cys180

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A ADE88156 3QVJ

1

10

20

. 3

70

Pseudomonas ﬂuorescens Pf0-1 Pseudomonas putida KT2440

50

60

90

120

130

140

GHAYSVVTTLDRTVPLIEDRLKLAGLYQRCASVRAS ATRFSIVTTLPRTLIIARHLLHQYGFHQHCAALHAI

150

160

C

170

GMAVLELEEDPVAAMEAIVRQAELAIREDKAEVICL DLPVLALEDGSGLAQEKVRERCIRALKEDGSGAIVL Cys180

180

ADE88156 3QVJ

Mycobacterium abscessus Mycobacterium vanbaalenii PYR-1

Cys79

ADE88156 3QVJ

190

200

210

GCGGMAGLDEQIRQRTGVPVVDGVTAAVTIAESLVR GCGGMATLAQQLTRELRVPVIDGVSAAVKMVESLVA

220

ADE88156 phenotype Species ortholog Pseudomonas fluorescens AllR+

230

240

LGLSTSKIRTYATPRPKKVIGWPGRLGR. LGLATSKHGDLAFPEKKALSGQFQSLNPF

Pseudomonas fluorescens

Pseudomonas tolaasi i 6264 + AllR

P. tolaasii

Pseudomonas aerugi nosa PAO1AllR

P. aeruginosa

Pseudomonas alcalAllR igenes -

P. alcaligenes

Pseudomonas pseudoal caligenesAllR

P. pseudoalcaligenes

Pseudomonas putidAllR a KT2440 +

P. putida

Pseudomonas stutzeri A1501 AllR

P. stutzeri

StenotrophomonasAllR maltophilia-D457

Cys184 ADE88156 3QVJ

Allantoicase

Streptosporangium roseum

100

VIQAGYGEHGREGLQELLNVPVVDITEAAASTAMFL HVIACFGDPGLLAARELAQGPVIGIAEAAMHMATMV

110

PuuE allantoinase

Rhodococcus opacus B4

80

ADE88156 3QVJ

AllB allantoinase

TPYFGAESVEGNFESYLAAIAVMDRVMA.YDQPFDA CPRAGVPSIEGHFDEAIAAVGVLEQIRAGREQGVDG Glu78

ADE88156 3QVJ

B Allantoin permease

40

ADE88156 3QVJ

30

.MRILVVNVNTTASITETIAEQARAVASPGTEIVGL SVRIQVINPNTSLAMTETIGAAARAVAAPGTEILAV

Page 24 of 28

Comamonas testosteroni CNB-1+ AllR Delftia acidovoransAllR SPH-1 -

+ Burkholderia cenocepaci a AU 1054 AllR

P. maltophilia

(Stenotrophomonas maltophilia)

P. testosteroni

(Comamonas testosteroni)

P. acidovorans

(Delftia acidovorans)

P. multivorans

(Burkholderia cenocepacia)

Figure 1 Bioinformatics evidence for the identification of P. fluorescens AllR. (A) Alignment of K. pneumoniae AllR (3QVJ) with the homologous P. flourescens protein (ADE88156). Known residues involved into the two-base catalysis are indicated by blue arrows; functionally equivalent residues identified in the PfAllR structure (see Figure 3D) are indicated by red arrows. (B) Examples of genetic associations of the putative PfAllR and similar genes (white boxes) with genes involved in purine catabolism (colored boxes). (C) Genotype/phenotype correlation between the presence of PfAllR orthologs in complete genomes and the AllR phenotype. The tree is in accordance with the NCBI taxonomy.


Page 25 of 28

2D Graph 1

Allantoin racemase E78D f = y0+a*x

Allantoin racemase Wt

B6

70 60

t

30

20 10

4

2 1

-10

0

20

230

240

250

260

10 0 -10 -20 -30 -40

t

-50 -60 -70 200

210

220

230

240

Wavelength (nm)

250

260

C

1

3 2 1

2

3

4

5

0

Allantoin (mM) 15

EnantiomericAllexcess (mM)

220

S-all utilizatio Regr_S-all R-all utilizatio Regr_R-all x column 6 v

0 0

210

-1

25

3

0

200

V0 (µM s ) -1 (mM s ) V 0

30

Y Data

40

S-allantoin Regr_S-all R-allantoin Regr_R-all

4

5 -1

CD (mdeg)

50

V0 (µM s ) -1 V (mM s ) 0

A

CD (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0.2 0.15 0.1 0.05 0 0.05 0.1 0.15 0.2

2

3

4

5

(S)

10

5

0 0 100

1

Allantoin (mM) [Allantoin] (mM)

[Allantoin] (mM)

(R) 100

80

60

200

300

400

40Time 20(sec)0 t_ts

X Data S-Allantoin excess vs tempo (min)

Figure 2. Racemase activity of P. fluorescens AllR. (A) Time course of S (red lines) and R (green lines) allantoin CD spectra in the presence of PfAllR (9 µg); spectra were taken every 60 s with 0.23 mM allantoin. (B) Kinetic analysis. Initial velocities were determined with different concentrations of S (○) or R (□) allantoin in the presence of 1.8 µg of wt PfAllR (left panel) or E78D mutant (right panel); data were fitted with the Michaelis-Menten equation. The solid red line in the left panel is a tentative fitting yielding a KM higher than the maximal concentration used in the assay. The initial slope of the hyperbola (dashed line) was used to estimate kcat /KM. (C) Catalyzed and uncatalyzed reaction courses; dashed lines represent the loss of CD signal in the absence of PfAllR (kuncat=6.2 x 10-5 s-1), circles are experimental data points in the presence of PfAllR, red and green lines are kinetic curves (55) calculated as a function of time, initial substrate concentration, Vmax (3.5 x 10-7 mM x kcat ), and KM.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

B

A

C

D T115

T119 T118

T114

V146

G181

G185

C180

C184

V150

2.7

4.3 Y76

3.5

F80

3.6 G75

C79

N9 V44 E78

N12

I47 D82

Figure 3. Crystal Structure of PfAllR. (A) Monomer in cartoon representation. (B) Dimeric organization in the cubic crystal form; the hexamer is generated by the crystallographic 3-fold axis. (C) Hexameric organization in the orthorhombic crystal form. (D) Comparison of the active site of PfAllR in the cubic crystal form (left, cyan carbon atoms) and KpAllR (right, orange carbon atoms; PDB ID: 3QVJ). Enolic allantoin (gray carbon atoms) was docked by superimposing the backbone chain of the two protein structures to the protein-substrate complex of KpAllR double mutant (PDB ID: 3QVK). Hydrogen bonded contacts are indicated by gray dashed lines. Distances of the putative catalytic dyads to the allantoin chiral center are reported in angstrom.


Page 27 of 28

H N

8 7

*

5

6

N H

O

3

4

HN 2

1

O

H N

H N

*

O

N H

H

O-

N H

H

O

N H

(S)-allantoin H N

-O

NH2

N H

C

O

D

NH2

C isotopic exchange H N

O

6

N H

4

N H

H

O-

2

0

Relative proton intensity

Relative peak ratio (C2 / C7)

13

Glu78 0

Gly181 N H

SH O

4

8

Cys180 12

Tyr76 NH

H N

O-

NH 20 2

16

N H OH

40

Velocity ( mM/min)

Velocity (mM/min)

ppm

160.0

159.5 ppm

0.20

H-C5 3

Time

N H

H

0

20

Glu78 40

O 60

Glu78

O

N H SH

N H

H

OGlu78

10

5.20

H proton exchange H N

100 120 140 160

NH2

N H

0.15 0.10

H N

-O

0.05NH2

H

O

N 10

0

H

20

30

N40 H

50

60

70

80

90

100

Time (min)

O

N

NH2

O O

N H

Tyr76 NH

Gly181 NH NH2

N H

H

OH

H N

OCys180

Glu78

N H S-

O

Cys180

O 0

(R

)-a

lla nt )-a oin + l l (S an w t )-a to in lla + n (R to w in )-a t + lla E7 nt (S o 8 in D )-a + lla E7 nt (R oi 8D )n al + la E7 nt 8Q oi n + E7 (S 8Q )-a lla nt (R )-a oin lla nt oi n

O

N H

N H OH Glu78

SH O

Cys180

Figure 4. Uncatalyzed and catalyzed allantoin racemization mechanisms. (A) Alternative mechanism of allantoin racemization. The labeled carbon atom in the (S)-allantoin molecule is indicated by an asterisk. (B) Examples of the time-dependent isotopic exchange between C2 and C7 (upper panel) and H-C5 proton-deuterium exchange (lower panel) as monitored by 13C and 1H NMR. (C) Kinetics of isotopic exchange between C2 and C7 of allantoin at pH 7.4 in the absence (empty circles) and in the presence (filled circles) of PfAllR. (D) Kinetics of C5 proton exchange in 98% D2O, pH 7.6 in the absence (right panel) and in the presence (left panel) of PfAllR. (E) Comparison of the activity towards the S and R enantiomers of allantoin (0.2 mM) for the wt and Glu 78 mutants (1.8 µg). (F) Proposed mechanism of allantoin racemization catalyzed by PfAllR.


N H

(R)-allantoin O

Time rac (min) vs Relative proton intensity x column 6 vs y column 6 H

SH O

O

O

0.20

O O

20

1

H N

NH2

5.25

N H

0.25

x column 5 vs y column 5 Time spont (min) vs Integrale spont (dati3)

O

2D Graph 1 f = a*exp(-b*x) 5.30 O f = a*exp(-b*x) ppm

0.00

Cys180

F

1

H N

H

Time (hr)

Cys180

30

80

N H

0.30

OH(S)-allantoin S-

0.05

OHN ppm 5.35

O

0.10

0.00

2

N H

N N O O N N H HH H H H

0.15

Time

159.0

H N

1 H Hexchange O proton O H N N NH2NH2 O O

0.25

O O

50

O

0.30

Time (hr)

E

1

O

2D Graph 1 f = a*exp(-b*x)

10

O

*

O

N H

N H

2D Graph 12 f=y0+a*exp(-b*x) f=y0+a*exp(-b*x)

8

H

NH2

O

N H

H

N H

O

O O

2

(R)-allantoin

H N

O NH2

3

NH2

O

N H

C2

O

Relative proton intensity

O NH2

O

C7

B

A

(S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

O

O-

-O

O-

-O

-

O

NH Biochemistry

H N

Page 28 of 28

2

-

OOC

NH3+

-OOC

O

NH3+ N H

O N H

1 O O O O YGCTSG F I SO TONL 2 O O OOC O O NH NH 3 OOC S 4 5 LSA VQM GYA LVA S T TS O O 6 2-methyl 2-phenylmalonate O O 7 O O S 8 9 10 2-methyl 2-phenylmalonate 11 I Q Y E CV G V H 12 O N NH O 13 N O N Plants 14 H H 15 I V I LG I LCTNTM D V 16 MO HyOd_race O OI I A m 17 as e_ OOC NH AA NH OOC 18 VI Kp Q9 G 3 Al lR 382 _3 H QV 19 O N J NH 20 O NH O O N O N 21 H H O O O O O 22 S AI I LG 23 O O L PN O O MAL A Fungi O O 24 OOC NH NH HYD OOC2-phenylmalonate 2-methyl 25 OOC NH OOC 26 ASP 27 O O 28 GLU O O O O 29 S O O 30 31 H O N NH 2-methyl 2-phenylmalonate 32 O 2-methyl 2-phenylmalonate 33 N O N H H 34 VI A V G Y 35 O O O O 36 OOC NH 37 NH OOC 38 39 O 40 O O O 41 O O Figure 5. Evolution of allantoin racemase within the Asp/Glu racemase superfamily. Unrooted neighborS 42 joining tree of the Asp/Glu racemase superfamily. Experimentally characterized proteins are indicated in bold 43 with PDB or Genbank accessions; other sequences are indicated with Genbank accessions followed by the 44 2-methyl 2-phenylmalonate taxid. Sequences from eukaryotic organisms (Plants, Fungi) are labeled. Tree branches assigned to functional 45 46 groups based on the phylogenetic analysis are shown in different colors next to sequence logos representing 47 residue conservation around the cysteine dyad (-3,+3). Reaction intermediates previously proposed based on 48 structural evidence are shown in colored boxes. 49 50 51 52 53 54 55 56 57 58 59 60 4

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The Structure and Function of a Microbial Allantoin Racemase Reveal

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