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Diatom allantoin synthase provides structural insights into natural fusion protein therapeutics Juntaek Oh, Anastasia Liuzzi, Luca Ronda, Marialaura Marchetti, Romina Corsini, Claudia Folli, Stefano Bettati, Sangkee Rhee, and Riccardo Percudani ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00404 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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ACS Chemical Biology

Diatom allantoin synthase provides structural insights into natural fusion protein therapeutics Juntaek Oha,§, Anastasia Liuzzib,§, Luca Rondac,d, Marialaura Marchettid, Romina Corsinib, Claudia Follie, Stefano Bettatic,d,f, Sangkee Rheea,*, Riccardo Percudanib,*

a

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea

b

Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma

Parco Area delle Scienze 23/A, 43124, Parma, Italy c

Department of Medicine and Surgery, University of Parma, 43124, Parma, Italy

d e

Biopharmanet-TEC Interdepartmental Center, University of Parma, 43124, Parma, Italy

Department of Food and Drug, University of Parma, 43124, Parma, Italy

f

National Institute of Biostructures and Biosystems, 00136, Rome, Italy

*For correspondence e-mail: [email protected]; [email protected] §

These authors contributed equally to this work.

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Abstract Humans have lost the ability to convert urate into the more soluble allantoin with the evolutionary inactivation of three enzymes of the uricolytic pathway. Restoration of this function through enzyme replacement therapy can treat severe hyperuricemia and Lesch-Nyhan disease. Through a genomic exploration of natural gene fusions, we found that plants and diatoms independently evolved a fusion protein (allantoin synthase) complementing two human pseudogenes. The 1.85 Åresolution crystal structure of allantoin synthase from the diatom Phaeodactylum tricornutum provides a rationale for the domain combinations observed in the metabolic pathway suggesting that quaternary structure is key to the evolutionary success of protein domain fusions. Polyethylene glycol (PEG) conjugation experiments indicate that a PEG-modified form of the natural fusion protein provides advantages over separate enzymes in terms of activity maintenance and manufacturing of the bioconjugate. These results suggest that the combination of different activities in a single molecular unit can simplify the production and chemical modification of recombinant proteins for multifunctional enzyme therapy.

Introduction (S)-Allantoin is the final product of purine degradation in nonhominoid mammals. Humans and the other apes accumulate poorly soluble urate as a final product (1). The conversion of urate into allantoin involves unstable intermediates 5-hydroxyisourate (HIU) and 2-oxo-4-hydroxy-4-carboxy5-ureidoimidazoline (OHCU) and three consecutive enzymes (2, 3): urate oxidase (Uox), HIU hydrolase (Urah), and OHCU decarboxylase (Urad). Inactivated copies (pseudogenes) of Uox, Urah, and Urad remain in our genome with elimination of the entire pathway (4–7). Although humans, at variance with other mammals (8), are evolutionarily adapted to high urate concentrations (9), excess of this metabolite in blood (hyperuricemia) results in pathological conditions that require medical treatment (10, 11). Hyperuricemia is commonly observed in the adult population as a consequence of environmental factors and genetic risk variants (12). It is also 2


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a complication of cancer chemotherapy as a consequence of tumor lysis (13). A rare, severe form of hyperuricemia and gout known as Lesch-Nyhan disease (LND) is observed in children affected by hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency (14). Enzymatic (uricolytic) therapy has been used for a long time to manage hyperuricemia when first line urate-lowering drugs are inadequate (15). Recombinant Uox is used in an unmodified form (rasburicase) for tumor lysis syndrome (13), or conjugated with polyethylene glycol (PEG) chains for extended half-life (pegloticase) to manage refractory gout (16). Uricolytic therapy is still not used in LND, although the current uricostatic therapy appears to be inadequate due to accumulation of upstream purines (17, 18). Long-term replacement of the lost uricolytic function would require the administration of chemically modified (PEGylated) Urah and Urad enzymes in addition to Uox —a solution enabling formation of the natural end product without accumulation of toxic intermediates of uricolysis (19, 20). Among the numerous challenges of a treatment involving multiple enzymatic activities are the cost and time of the production and chemical modification of different recombinant proteins. These problems can be alleviated by combining different activities in a single molecular entity (21). The combination of functionally associated domains in the same protein chain is a solution often exploited by nature (22, 23), and genes encoding fused domains of the uricolytic pathway are indeed found in several genomes. Fused Uox and Urad domains (steps 1 and 3) are found in certain bacilli, and fused Urah and Urad domains (steps 2 and 3) are found in both prokaryotes and eukaryotes. Bidomain Urad-Urah proteins were first identified in plant peroxisomes (24). The protein from Arabidopsis thaliana was found to be able to catalyze the second and third steps of the uricolytic pathway (25, 26) and named allantoin synthase. The structure of the truncated Urad domain of A. thaliana has been obtained (27), and the structure of isolated Urah domains is known in other organisms (28–31). However, no information on the structural organization of the two fused domains is available. Here we show that diatoms (e.g. Thalassiosira pseudonana, Phaeodactylum tricornutum) and other planktonic algae (e.g. Emiliania huxleyi) have evolved independently from 3



plants a Urad-Urah gene fusion that functions as allantoin synthase. We describe the structure and function of the fusion protein from P. tricornutum and show through PEGylation experiments that this enzyme is potentially suitable for therapeutic applications.

Results and Discussion Urad-Urah gene fusions Using a sensitive protein domain search, we identified the presence of Urah and Urad domains in eukaryotic genomes (Fig 1). Consistent with previous observations (3), genes encoding Urah and Urad proteins tend to occur in pairs (i.e., they are present together or absent together in a given genome). These two domains are found in all five eukaryotic supergroups (32, 33) except Excavata. In many groups of organisms such as Metazoa, Fungi, and Amoebozoa, they are present as separated proteins. However, they are fused in a single bidomain protein in Viridiplantae (including green algae and land plants) as well as in Stramenopiles and Haptophyte algae (Fig 1A). Reconstruction of separated and concatenated domain phylogenies provides evidence that the bidomain proteins of Viridiplantae and Stramenopiles/Haptophyte have distinct origins (Fig 1B and Fig S1). A similar search in prokaryotic genomes revealed the frequent presence of bidomain Urad-Urah proteins in Proteobacteria (•, •, and • subdivisions) and Terrabacteria (Actinobacteria and Firmicutes) (Fig S2). In accordance with protein domain phylogenies, multiple gene fusion events likely also occurred in prokaryotes. Bacterial and eukaryotic proteins are not monophyletic, suggesting in particular the occurrence of horizontal gene transfer among alphaproteobacteria and Stramenopiles/Haptophyte ancestors (Fig S3). Although deriving from several independent gene fusions, all these bidomain proteins are composed of a N-terminal Urad and a C-terminal Urah domain, consistent with the requirement of a free Urah C terminus for catalysis (29). The linker region between Urad and Urah is highly variable in

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eukaryotic proteins (Fig 1C). Plant proteins possess large linkers of 30-50 amino acids enriched in Ala (15%), Ser (11%), Pro (10%), and Gly (8%). A conserved motif corresponding to the consensus (R- [LIVM]-X (5)- [HQ]-L) of the type 2 peroxisomal targeting sequence (PTS2) is involved in import of the protein into peroxisomes (24, 25). By contrast, Stramenopiles/Haptophyte proteins, similarly to bacteria, possess short linkers devoid of PTS2 signals (Fig 1C and Fig S2). No targeting signals can be identified in other regions of these proteins. This is at variance with Urah and Urad proteins in other eukaryotes which typically possess PTS2 signals at the N terminus (Urah) or PTS1 signals at the C terminus (Urad).

Activity of the Urad-Urah protein from P. tricornutum To gain insights into the function and structural organization of bidomain Urad-Urah proteins, we recombinantly expressed in E. coli genes from different eukaryotic and prokaryotic sources. In some cases we obtained either an insoluble protein (Magnetospirillum magnetotacticum) or proteins prone to hydrolytic cleavage (Arabidopsis thaliana, Bradyrhizobium japonicum). However, the expression of a cDNA clone from the diatom Phaeodactylum tricornutum (34) produced a stable and active protein that was also amenable to crystallization (see below). The cloned P. tricornutum cDNA corresponds to a gene (PHATRDRAFT_49522) located on the chromosome 22 (501852..502849, complementary strand). The gene is interrupted by a short intron located near to the boundaries of the Urad and Urah domains (Fig S4). The encoded protein of 299 aa is defined as ‘hypothetical’ in Genbank, but is predicted to be involved in uricolysis in DiatomCyc (35). A recombinant protein of the expected size (~35 kDa for the monomer) was produced in E. coli and purified to apparent homogeneity by affinity column chromatography (final yield 14 mg/L). The P. tricornutum protein is distantly related to the experimentally validated homolog from A. thaliana (24% identity; see also Fig 1B). However, kinetic characterization provided evidence for its activity in two consecutive reactions of the uricolytic pathway leading to the stereoselective formation of (S)-allantoin (Fig 2 and Fig S5). This protein will be hereafter referred to as P. tricornutum allantoin 5



synthase (PtAlls). The separate monitoring of the two activities (Fig 2A,B) revealed values similar to those previously measured for the separated enzymes from Danio rerio (20), with specific activities of 400.8 ± 20 and 99.4 ± 0.3 µmol mg-1 min-1 for the Urah and Urad reactions, respectively. In the presence of the urate substrate and excess of Uox, accumulation of OHCU during the PtAlls reaction was observed through circular dichroism spectroscopy (Fig 2C-E). Furthermore, in these conditions, a substantial increase of the velocity of OHCU degradation can be observed by adding to the reaction a monodomain Urad protein (Fig 2F), suggesting that the unstable OHCU intermediate is released in solution.

Crystal structure of allantoin synthase X-ray crystallographic analysis at 1.85 Å-resolution (Table S1) revealed that PtAlls folds in two distinct domains (Fig 3). In the monomer, the N-terminal •-helical and the C-terminal •-sandwich domains are arranged in an orthogonal direction along the long axis of each domain, almost forming an L-shaped molecular architecture (Fig 3A). The •-helical (residues 1–174) and •-sandwich (residues 181–299) regions correspond to monomeric structures of Urad (27, 36, 37) and Urah (28– 31), respectively (Fig S6), with sequence identities of 20% and 45%, respectively, and a root-mean square deviation of 1.9–2.0 Å for 125 C• atoms and 1.0–1.1 Å for 107 C• atoms. In particular, residues Cys79 to Gln102 for Urad domain in PtAlls are highly disordered and not modeled. Under our crystallization conditions, there are four monomers of PtAlls in the asymmetric unit, indicating that each PtAlls is in a different crystallographic packing environment. Therefore, there are some variations in the quality of electron density map among four monomers. However, the overall structures of four PtAlls are essentially identical, with a root-mean square deviation of 0.21– 0.67 Å for 270–272 Cα atoms. The presence of four monomers in the asymmetric unit is consistent with the elution profile from the size-exclusion chromatography (Fig S7). Two monomers dimerize through interactions of the Urah β-strands, generating a layer with a central β-sandwich flanked by

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two separated α-helical domains (Fig 3B). As in monodomain Urah and transthyretin (38), a functional tetramer is formed by the superposition of two dimeric layers related by a two-fold symmetry. In the PtAlls tetramer, monomers are packed in a 222-fold symmetric manner, forming the catalytically competent configuration of Urah at the structure core (Fig 3C). An orthogonal location of Urad domain relative to Urah also constrains its location in a 222-fold symmetric way, but on the surface of the Urah tetramer. Urad domains from different layers constitute a dimer and each dimer is located at opposite ends of the Urah tetramer (Fig 3C,D). The homodimeric conformation of Urad domain is like that observed in the truncated protein of A. thaliana (27) and different from that observed in monodomain Urad (36, 37). Dimeric packing of PtAlls within one layer buried about 2,000 Å2 of surface area (Fig 3B) and an additional area of 900 Å2 is also buried by tetramerization (Fig 3C). Due to an L-shaped arrangement of Urad and Urah domains, there are no direct interactions between Urad and Urah domains within a monomer except for the linker region. This exposed region is well defined in the electron density map, indicating a rigid conformation (Fig 4A,D). Extensive interfaces in a tetrameric 222-symmetric packing result in various inter-subunit interactions. The inter-subunit interactions between Urad and Urah are newly characterized in PtAlls. Specifically, anti-parallel α7Urad and α8Urad mediates hydrophobic interactions with Urah domain from the adjacent subunit in the same layer, mainly with α9Urah and a following loop region (Fig 4B,D). These extensive interactions - corresponding to a buried surface area of 1,330 Å2 contribute to stabilization of a bifunctional allantoin synthase. The interfaces among Urad domains involving helices α8 and α5 seal the borders of the PtAlls tetramer through hydrophobic interactions (Fig 4C,E). Despite the overall similarity with the A. thaliana dimer, interacting residues are not conserved (Fig S6). The active sites of monodomain Urad and Urah were characterized in previous studies (27, 28, 30, 36, 37), including the substrate binding sites and functional roles of active site residues. Those active sites in PtAlls are well conserved in both structure and sequence: His75, Glu95 (not

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modeled), Arg169 in Urad, and His186, Arg225, His283 in Urah (Fig S6). A total of four Urad active sites are located within each PtAlls tetramer, and two independent Urah active sites are located at the center of the inter-layer interface (Fig 4D,E). The Urad-Urah active sites are about 40 Å apart. Their entrances are 90-degrees away from each other and completely separated by several layers of structural elements. Therefore, this architecture precludes any possible substrate channeling from Urah to Urad for consecutive enzyme reactions.

PtAlls PEGylation Two PEGylated PtAlls were prepared exploiting different conjugating chemistries (Fig 5): 20 kDa maleimido-functionalized PEG (MAL-PEG) was used to derivatize cysteine side chains as recently reported for DrUox, DrUrah and DrUrad PEGylation (20), while 10 kDa N-hydroxysuccinimide PEG (NHS-PEG) was used to derivatize lysine side chains, similarly to pegloticase (16). In the PtAlls structure, 12 thiol groups and 48 ε-amino groups are exposed on the protein surface (Supplemetary Fig S8). SDS-PAGE of PEGylated and unmodified PtAlls showed a higher average number of conjugated NHS-PEG chains per PtAlls monomer, with respect to MAL-PEG derivatized protein (Fig 5A,B). Starting from the degree of monomer derivatization, the percentage of unmodified tetramers can be calculated through combinatorial analysis, as previously reported for PEGylated uricolytic enzymes 20. For PtAlls, the measured small amounts of non-PEGylated monomers correspond to a very low calculated population of unmodified (unprotected) tetramers (0.19% and 0.07%, for the enzyme derivatized with MAL-PEG and NHS-PEG, respectively). . The comparison of catalytic efficiency, as expressed by kcat/KM values, for the Urah and Urad reactions with PEGylated and unmodified PtAlls revealed reduced Urad efficiency in the MALPEG conjugate and increased Urah efficiency in both conjugates (Fig 5C,D and Fig S9 and Table S2). Urate degradation by PtAlls PEGylated with MAL-PEG or NHS-PEG in the presence of PEGylated DrUOx was followed by circular dichroism (Fig 5E,F). The decreased efficiency of MAL-PEG PtAlls in the Urad reaction caused accumulation of the OHCU intermediate (Fig 5E), 8


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while PtAlls PEGylated with NHS-PEG retained full Urad catalytic efficiency and brought to complete conversion of urate into (S)-allantoin without intermediate accumulation, similarly to the unmodified enzyme (Fig 5F and Fig S5).

Conclusions A diatom gene fusion encoding a bifunctional allantoin synthase could simplify the manufacture of a PEGylated preparation for uricolysis by replacing two separated enzymes. There are several examples of fusion protein therapeutics obtained by genetic engineering (21). Our results point to the exploitation of natural gene fusions for enzyme therapeutics requiring multiple activities. Eukaryotes possessing allantoin synthase (Viridiplantae, Stramenopiles, and Haptophyte) are united by the presence of a photosynthetic organelle which is thought to originate from a cyanobacterium through a single primary endosymbiotic event followed by secondary symbioses (39). While this suggests the possibility of a common origin, the bidomain proteins of Viridiplantae and Stramenopiles/Haptophyte derive from independent gene fusions (see Fig 1B and Figs. S1,S3). Neither of the two groups of eukaryotic proteins has close homologs in cyanobacteria, and bidomain Urad-Urah proteins are not identified in the available cyanobacterial genomes (see Fig S2). The reason for the parallel evolution of allantoin synthase in major photosynthetic eukaryotic lineages is possibly related to the importance in these organisms of allantoin and purine degradation for the metabolism of growth-limiting nitrogen. In many plant species, allantoin is used for nitrogen storage and translocation (40). As structural and functional evidence demonstrates absence of substrate channeling, the selective advantage of Urad-Urah fusions could be that of ensuring coexpression and colocalization of the two domains. The internal PTS2 in plant sequences targets the joint domains to peroxisomal matrix, where Uox is also localized (24). The absence of this signal in PtAlls is consistent with the loss of PTS2-mediated import in diatoms (41). A targeting signal is also lacking in P. tricornutum Uox (PHATRDRAFT_29736), suggesting relocation of the pathway in diatoms. Absence of the internal 9



targeting sequence and presence of a short, rigid linker between the two domains (see Fig 4) could be among the reasons for the higher stability of the diatom bidomain protein. A possible disadvantage of the Urad-Urah fusion is the inability to vary the stoichiometry of the two domains. Formation of the natural stereoisomer of allantoin depends on Urad catalysis and avoidance of OHCU spontaneous decay (which produces a racemic mixture). However, PtAlls is much more efficient in catalyzing the first reaction than the second (see Table S2). A possible explanation for the lack of adaptation of the kinetics parameters in allantoin synthase is that urate oxidation by Uox is the limiting step; this prevents accumulation of OHCU unless Uox is present in molar excess (see Fig 2D,E). PtAlls was conjugated with PEG, a biocompatible polymer that improves rheological properties and bioavailability of protein therapeutics (42–45). Two alternative protocols yield PtAlls tetramers derivatized with a relatively high average number of conjugated PEG chains (lysine PEGylation) or a smaller number of longer chain PEG (cysteine PEGylation). Both protocols allowed good retention of Urad catalytic efficiency and increased Urah efficiency with respect to the unmodified protein. The latter effect is due to PEGylation affecting both kcat and KM. On the other hand, the slightly reduced efficiency observed for the Urad activity of MAL-PEG conjugates is mainly related to an effect on kcat (see Table S2). It has to be noted that cysteine PEGylation of the isolated DrUrad domain led to enzyme inactivation, while lysine PEGylation (through the 2-iminothilane extension arm) led to a reduced activity (20). Either the use of a combination of separated Urah and Urad enzymes or the bifunctional protein described here would represent an improvement of the current standard of care based on the administration of Uox alone. Even though in the absence of substrate channeling this fusion protein does not provide kinetics advantages over separated enzymes, its use in therapeutic applications has advantages both in terms of ease of protein production and chemical modification and in terms of activity maintenance in the bioconjugate. Our structural analyses reveal that (S)-allantoin synthase catalyzes its two independent, consecutive reactions by forming a tetrameric conformation for Urah in the center of the enzyme and two Urad 10


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homodimers on the surface of the enzyme. This molecular architecture answers the question of how tetrameric Urah and dimeric Urad characterized in the previous studies could be accommodated in a bifunctional enzyme. Interestingly, this architecture also provides the possibility to predict the organization of other proteins containing fused domains involved in purine catabolism (Fig S10). These fusion proteins can be modeled as a central tetramer flanked by two homodimers or as a central hexamer flanked by three homodimers. By contrast, other domain fusions that would be expected from a biochemical standpoint, but cannot be modeled in a similar architecture are not observed. In particular, a gene fusion involving tetrameric Uox and Urah domains (steps 1 and 2) could not have the possibility of producing a functional quaternary structure. Together with the observation of multiple independent origins for Urad-Urah proteins, this analysis suggests that quaternary structure is key to the evolutionary success of protein domain fusions. Along these lines, synthesis of an artificial multidomain protein such as a trivalent enzyme with Uox, Urah, and Urad activities -the most desirable target for uricolytic therapy- entails the design of a molecular architecture compatible with the quaternary structure of these domains.

Methods Bioinformatics —The search for Urad and Urah proteins (Table S3) was conducted at the NCBI using the A. thaliana bidomain protein as a query and the DELTA-BLAST algorithm (46). Multiple alignments of bidomain and monodomain proteins were conducted with the local algorithm implemented in DIALIGN-TX (47). Multiple alignments of concatenated domains were conducted with CLUSTALW (48). The bacterial tree was constructed using the bp_taxonomy2tree.pl script of Bioperl (49). The eukaryotic tree was based on a chronogram obtained through the TimeTree web server (50); the chronogram was edited with TreeGraph (51) to reflect proposed relationships among eukaryotic supergroups (32). Protein phylogenetic trees of separated and concatenated domains were constructed using maximum-likelihood and branch support methods implemented in the PhyML web server (52, 53).

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Cloning, expression and purification of PtAlls —The PtAlls coding sequence (XM_002184228.1) was amplified from total P. tricornutum cDNA (kindly provided by A. Falciatore, CNRS Université Pierre et Marie Curie, Paris) using an NdeI-tailed upstream primer (5’CATATGTCCGCTCCTGTAACC-3’) and a BamHI-tailed downstream primer (5’GGATCCTCTCTACGACCCGCGG-3’). The PCR product (915 bp) was cloned into pET28b in frame with N-terminal His6-tag. For crystallization the PtAlls coding sequence was cloned in a modified pET28b vector (Merck) containing sequences for the His5-tagged maltose-binding protein (His-MBP) and TEV protease recognition site. The recombinant plasmids were transferred into E. coli BL21 (DE3) cells (Novagen) for protein over-expression. E. coli cells were incubated at 37°C until an optical density at 600 nm of 0.6-0.8. After addition of 0.5 mM IPTG, E. coli cells were cultured for 14-16 hours at 20°C. The cells were sonicated and centrifuged in Buffer A containing 50 mM HEPES pH 7.5 and 150 mM NaCl. Cell lysate was loaded to a HisTrap HP column (GE Healthcare). The PtAlls was eluted with Buffer A plus 300 mM imidazole and the His-MBP tag was removed by dialyzing against Buffer A with 2 mM DTT overnight at 4°C, with a 20:1 molar ratio of PtAlls to TEV protease. An additional round of affinity chromatography and size-exclusion chromatography using Superdex-200 (GE Healthcare) with Buffer A was performed to purify tagfree PtAlls. For biochemical characterization and PEGylation, the His-tagged PtAlls version was purified by affinity chromatography on Talon resin (Clontech) using 50 mM Tris buffer pH 8.0 containing 300 mM NaCl. Before PEGylation, the protein was further purified by anionic exchange on a 5 ml Q FF column (GE Healthcare) in 50 mM Tris, pH 8.0, and eluted with a NaCl gradient. Protein fractions were analyzed on SDS-PAGE, pooled, and diafiltered in 100 mM potassium phosphate, 150 mM NaCl, pH 7.4. Enzyme activity determination —Urah and Urad activities of PtAlls were measured separately by monitoring the absorbance signal at 312 nm and 257 nm, respectively. HIU and OHCU were produced in situ from urate during each measurement by adding DrUox, or DrUox and DrUrah. Spectrophotometric measurements were conducted at room temperature in a 1 cm path-length

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cuvette with V-750 UV-visible Jasco Spectrophotometer, using a 0.1 mM urate solution, in 100 mM potassium phosphate pH 7.4 buffer. Kinetic parameters of native and PEGylated proteins were determined at 37 °C in the presence of different concentrations of HIU or OHCU substrate in 100 mM potassium phosphate, pH 7.4, with a Cary 4000 (Varian) spectrophotometer equipped with a thermostated cell holder. Circular dichroism —Circular dichroism spectra were recorded in 20 mM potassium phosphate, pH 7.4 with a Jasco J715 spectropolarimeter equipped with a Peltier thermostatic cell, using a 0.2 cm path-length quartz cuvette. Scanning kinetics measurements were carried out between 200 nm and 340 nm, at 2 min intervals, in the presence of 200 µM urate, 0.22 µM DrUox and 0.15 µM PtAlls, at 37 °C. Time-course measurements at 257 nm in presence of different DrUox:PtAlls ratio were performed at room temperature using a 1 cm path-length quartz cuvette. The maintenance of secondary structure in PEGylated proteins was verified by spectra collected in the far-UV region between 195 nm and 260 nm on 2 µM (monomer concentration) protein solutions, at 20 °C. Crystallization, data collection and structure determination —Purified PtAlls was concentrated to 10 mg/ml for crystallization. Crystals were then obtained by sitting drop vapor diffusion method at 22 °C by mixing equal volumes of protein solution and reservoir solution containing 0.2 M MgCl2, 0.1 M HEPES (pH 7.0) and 20 % (w/v) PEG 6000. X-ray diffraction data were collected at 100 K on beamline 7A at the Pohang Accelerator Laboratory (Korea). Ethylene glycol (20 %, v/v) was used as the cryo-protectant and data processing was carried out by using iMosflm (54).The space group of PtAlls crystals is P21, with four monomers in the asymmetric unit (Table S1). The 1.85 Åresolution structure of PtAlls was determined by molecular replacement program in PHENIX (55)⁠ by using Urah structure from Brucella melitensis (PDB id: 4Q14) as a search model. Initial model was further built by Buccaneer (56)⁠ , and manual building and refinement was conducted for several cycles by using the programs PHENIX and COOT (57) PEGylation —Solutions containing 10 mM PEG (20 kDa MW) functionalized with maleimido– group (MAL-PEG) or 40 mM PEG (10 kDa MW) functionalized with N-hydroxysuccinimide

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(NHS-PEG) were freshly prepared and added to 1.45 mg/ml aliquots of PtAlls in 100 mM potassium phosphate, 150 mM NaCl, pH 7.4, and then incubated at 20 °C for 30 minutes. Reactions were carried out at a molar ratio of 7.5/1 Mal-PEG/cysteine and 6/1 NHS-PEG/lysine and quenched with a 6-fold excess PEG over free cysteine and lysine side chains, respectively. To remove excess reagents, samples were diafiltered at 4 °C in 100 mM potassium phosphate, 150 mM NaCl, pH 7.4, using Amicon Ultra-0.5 ml centrifugal filter devices (Merck-Millipore) with 100 kDa cut-off. For SDS-PAGE, 6 µg of the unmodified and PEGylated enzymes (protein weight) were precipitated in acetone (1:5 v/v ratio). The pellets were dried and resuspended in sample buffer. Gel was stained with Biosafe® Coomassie (Biorad) for protein detection and scanned on a ChemiDoc imager (Biorad). Relative band intensities were calculated by densitometric analysis by ImageLab software (Biorad). PEGylated monomers of PtAlls were detected after 10 minutes of incubation with a 5% BaCl2 solution, followed by staining with a 0.05 M I2 solution.

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Accession numbers The atomic coordinates and structure factors of allantoin synthase have been deposited in the Protein Data Bank with accession code 5Z5M.

Authors contributions J.O. and A.L. contributed equally to this work. J.O. performed cloning, protein expression and purification, crystallization, data collection and, together with S.R., structure determination. S.R., J.O., and A.L. performed structural analysis. A.L. performed cloning, protein expression and purification, and biochemical experiments. L.R. and M.M. performed PEGylation and characterization of bioconjugates. R.C. and C.F. performed cloning, protein expression and purification. R.P., S. B., M.M. and L.R. performed data analysis. R.P. conceived the study. R.P., S.R., and S.B. designed the experimental procedures and wrote the manuscript.

Acknowledgements We thank A. Falciatore for the kind gift of P. tricornutum cDNA, and C. Bowler and R. Berni for discussion.

Funding Telethon grant GGP13149 “Development of an uricolytic treatment for HPRT deficiency in animal models” (to R.P.) Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ01325801), Rural Development Administration, Republic of Korea (to S.R.).

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Figure legends

Figure 1. Urad-Urah gene fusions in eukaryotes. (a) Occurrence of Urad and Urah genes in eukaryotic genomes. The tree represents a consensus phylogeny of eukaryotes (32, 33), with nodes corresponding to the main eukaryotic supergroups labeled in capital letters. Grey branches represent proposed relationships among supergroups and among SAR (Stramenopiles, Alveolata, Rhizaria) and Haptophyta (32, 59). Grey nodes correspond to unresolved relationships in NCBI taxonomy. Relationships within groups and divergence times are from TimeTree (50). (b) Maximum-likelihood phylogeny of concatenated Urad and Urah domains; nodes with >0.9 support based on non parametric approximate likelihood ratio test (53) are indicated by black circles. (c) Linker region in aligned bidomain Urad-Urah proteins. Figure 2. Bifunctional activity of PtAlls. (a) Decrease of absorbance of the HIU signal with the addition of 14 µM PtAlls (arrow); HIU was generated in situ using 1 µM DrUox and 0.1 mM urate. (b) Decrease of absorbance of the OHCU signal with the addition of 28 µM PtAlls (arrow); OHCU was generated in situ using 1 µM DrUox and 0.07 µM DrUrah and 0.1 mM urate. (c) Reference CD spectra of the natural stereoisomers (S)HIU (green line) and (S)-OHCU (blue line); a Cotton effect for HIU is observed around 257 nm, indicated on the graph with a dashed line. Y-axis units are in molar ellipticity ([θ]). (d) OHCU accumulation measured at 257 nm at increasing Uox:PtAlls concentration ratios in reactions with 0.2 mM urate. (e) Dependence of OHCU accumulation on the relative concentrations of Uox and PtAlls. (f) Kinetics of OHCU decarboxylation catalyzed by PtAlls in the absence (solid line) and in the presence (dashed line) of excess DrUrad. Figure 3. Ternary and quaternary structure of PtAlls. (a) An L-shaped molecular architecture of monomeric PtAlls is shown with secondary structure elements. (b) Dimeric layer, with two monomers related by the non-crystallographic two-fold symmetry in different colors. (c) View of the functional PtAlls tetramer obtained by a 90-degrees rotation of B along the horizontal axis with additional layer. (d) The dimeric organization of Urad domain is shown with a 90-degrees rotation of c along the vertical axis. Figure 4. Structural rationale for PtAlls activity and stability. (a) Linker region (in stick) with the electron density map for aa 175-180 shown at 1.5σ contour level. (b) Detail of the Urad-Urah inter-subunit interface involving interaction of helices α9Urah and α7Urad. (c) Urad-Urad inter-subunit interface involving interaction of helices α8 and α5; intrasubunit π-interactions are indicated by grey dashes. (d) Semi-transparent surface representation of 16


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the tetramer with ligands at the active sites; ligands were positioned by superposition with Urad structure (PDB id: 2Q37) in complex with (S)-allantoin (black spheres) (27) and Urah structure (PDB id: 2H0F) in complex with 8-azaxanthine (blue spheres) (28); orientation and colors are as in Fig 3c. (e) A 90-degrees rotation of D along the vertical axis. Figure 5. Characterization of PEG-conjugated PtAlls. (a-b) SDS-PAGE of unmodified and PEGylated PtAlls after Biosafe® Coomassie (a) and PEGspecific barium iodide dye (b) staining. Lane M, molecular weight standards; lane 1, unmodified PtAlls; lane 2, MAL-PEG PtAlls; lane 3, NHS-PEG PtAlls. (c-d) Urah (c) and Urad (d) catalytic efficiency of unmodified (black), MAL-PEG (red) and NHS-PEG (blue) PtAlls. *p < 0.05 versus unmodified PtAlls. (e-f) Time-evolution of circular dichroism spectra of solutions containing MALPEG (e) or NHS-PEG (f) in the presence of PEGylated DrUox and 200 µM urate, 20 mM potassium phosphate, pH 7.4, at 37 °C. Spectra were acquired every 2 minutes.

17



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Diatom fusion protein

G PE

Uox

Urate

Urad

Urah

HIU

OHCU

(S)-allantoin


A.a nop hag effe ren s Chrysochrom ulina.sp.

b

se

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a

M .c om m od

P.pa ra

ula cic fas M. o D.reri

ris

A.a no ph ag eff ere ns Chrysoc hromulin a.sp.

s

C.crispus

sa

an m

ras

or of

sitic a

N.c

e .n

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

ACS Chemical Biology ud

p Homo sapiens T. Homo sapiens Homo sapiens Macaca fascicularis Macaca fascicularis Macaca fascicularis MetazoaDanio rerio Danio rerio Danio rerio OPISTOKONTA D.d Cryptococcus neoformans isc oid Cryptococcus neoformansneoformans Cryptococcus tiva eum sa Neurospora crassa Fungi O. A.thaliana NeurosporaNeurospora crassa crassa Dictyostelium discoideum Dictyostelium discoideumdiscoideum Dictyostelium C.rein Entamoeba histolytica AMOEBOZOA V.c hardti i ar EntamoebaEntamoeba histolytica histolytica ter Naegleria gruberi 0.6 i 0.6 >0.9 Naegleria gruberi Naegleria gruberi Trypanosoma brucei EXCAVATA Trypanosoma brucei Trypanosoma brucei Oryza sativa E.huxleyi Oryza sativaOryza sativa Arabidopsis thaliana ArabidopsisArabidopsis thaliana thaliana Viridiplantae Volvox carteri tum ornu a Volvox carteri Volvox carteri P.tric an Chlamydomonas reinhardtii on ud Chlamydomonas e Chlamydomonas reinhardtii reinhardtii ps Micromonas commoda T. MicromonasMicromonas commoda commoda Chondrus crispus ARCHAEOPLASTIDA Chondrus crispus Chondrus crispus Phaeodactylum tricornutum Phaeodactylum Phaeodactylum tricornutumtricornutum Thalassiosira pseudonana Thalassiosira Thalassiosira pseudonanapseudonana Aureococcus anophageffer. Aureococcus anophageffer. Aureococcus anophageffer. URAD URAH Linker region StramenopilaPhytophthora parasitica Phytophthora 160 170 180 190 Phytophthora parasitica parasitica SAR Plasmodium falciparum1 XP_001699572.1 R L S G L F A L P P D A . . . A D R A A R . . . . . . . . . R A E Q V L T H L A P G H . . . . . . . . . . G G A . . P L R S P I T T H XP_002947784.1 R L C G L F G I S D Y V E S V A A R T Q R . . . . . . . . . R A E Q V L T H L A P A P . . . . . . . . . . G G . . . P L R S P I T T H PlasmodiumPlasmodium falciparum falciparum EEC75394.1 RLAKLFASEPVAPPSSTVGGPTSQSDKAADRMRIIGAHLGSHTQHSANKAPEITGSSNRTRPPITTH Bigelowiella natans NP_200630.1 R M A K L F S D K A K V I . S E T D S S S S P V S T K P Q D R L R I0.6 IGGHLNVAAEAKAPK.........RSRPPITTH BigelowiellaBigelowiella natans 2 natans XP_002184264.1 R L L S K I D T S D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Q G F L T C H Emiliania huxleyi XP_002288652.1 R L L D K V D Y A E G A N G H G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G F L T C H Emiliania huxleyi XP_009037636.1 R L L D A V A H T P A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G F L T C H Emiliania huxleyi R L N EPlus L V R P NEnvironment PT..................................................GFLTCH Haptophyta Chrysochromulina sp. 3 XP_005768231.1 ACS Paragon RLRALVSPNPT..................................................GFLTCH Chrysochromulina sp. Chrysochromulina sp. KOO25041.1 100 My PTS2

URAD URAH Pseudogene

C

a

is lar icu sc .fa M rio D.re

tum a an on

rnu

ico P.tr

c

C.r. V.c. O.s. A.t. P.t. T.p. A.a. E.h. C.sp.

100.0

100.0

100.0

Figure 1. Urad-Urah gene fusions in eukaryotes. (a) Occurrence of Urad and Urah genes in eukaryotic genomes. The tree represents a consensus phylogeny of eukaryotes 32,33, with nodes corresponding to the main eukaryotic supergroups labeled in capital letters. Grey branches represent proposed relationships among supergroups and among SAR (Stramenopiles, Alveolata, Rhizaria) and Haptophyta 32,58. Grey nodes correspond to unresolved relationships in NCBI taxonomy. Relationships within groups and divergence times are from TimeTree 50 . (b) Maximum-likelihood phylogeny of concatenated Urad and Urah domains; nodes with >0.9 support based on non parametric approximate likelihood ratio test 53 are indicated by black circles. (c) Linker region in aligned bidomain Urad-Urah proteins.

Page 27 of 30


aa

0

20

40

60

80

Time (s)

100 120 140

O

0.6

H N

0.4

O

Urate

OOC OH H N

H2O

NH2

O

N H

N

O

O

N

N

(S)-5-hydroxyisourate

-20000 O OH H N

-40000

N H

N

O

OH H N

HN

O

CO2-

NH2

N

OOC OH H N

H2O

O

N

(S)-5-hydroxyisourate

240

260

O

CO2-

NH2

O

NH2

N N H (S)-OHCU

O

H N O

O O

N N H (S)-OHCU

280

300

N H N H H (S)-allantoin

320

340

Wavelength (nm) 20

40

60

80 100 120 140

Time (s)

d d

0.5 0.4 0.3

ee 0 -10 1:1 10:1 20:1 40:1

-20 -30

0.2

0

0.1 0.0 0

20

40

60

80

Time (s)

100 120

60 50

100 200 300 400 500 600

Time (s)

% OHCU

0

40 30 20 10 0 1

10

[Uox]/[PtAlls]

H N

O

0

0.2 0.0

ff

O

O H2O2

Urate

HN

O

O2 H2O

H N

O

20000

H2O2

θ257nm (mdeg)

Abs257nm

HN

O2 H2O

O HN

[θ] (deg cm2 dmol-1)

Abs312nm

40000

0.4

0.0

b b

257nm

0.6

0.2

Abs257nm

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

cc

0.8

100


Figure 2. Bifunctional activity of PtAlls. (a) Decrease of absorbance of the HIU signal with the addition of 14 μM PtAlls (arrow); HIU was generated in situ using 1 μM DrUox and 0.1 mM urate. (b) Decrease of absorbance of the OHCU signal with the addition of 28 μM PtAlls (arrow); OHCU was generated in situ using 1 μM DrUox and 0.07 μM DrUrah and 0.1 mM urate. (c) Reference CD spectra of the natural stereoisomers (S)-HIU (green line) and (S)-OHCU (blue line); a Cotton effect for HIU is observed around 257 nm, indicated on the graph with a dashed line. Y-axis units are in molar ellipticity ([θ]). (d) OHCU accumulation measured at 257 nm at increasing Uox:PtAlls concentration ratios in reactions with 0.2 mM urate. (e) Dependence of OHCU accumulation on the relative concentrations of Uox and PtAlls. (f) Kinetics of OHCU decarboxylation catalyzed by PtAlls in the absence (solid line) and in the presence (dashed line) of excess DrUrad.

N H N H H (S)-allantoin

O



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aa

bb

cc

α7

Asp175

His137

Phe155

α8

Thr138 Ala141

α8

Glu251

Gln180

Ala254

Gly145

Lys65*

α9 β1

Met156

Met156*

Glu152*

Phe155*

α5 Ser255

α5

Lys65

Glu152

α8

dd

e e

90°


Figure 4. Structural rationale for PtAlls activity and stability. (a) Linker region (in stick) with the electron density map for aa 175-180 shown at 1.5σ contour level. (b) Detail of the Urad-Urah inter-subunit interface involving interaction of helices α9Urah and α7Urad. (c) Urad-Urad inter-subunit interface involving interaction of helices α8 and α5; intrasubunit π-interactions are indicated by grey dashes. (d) Semi-transparent surface representation of the tetramer with ligands at the active sites; ligands were positioned by superposition with Urad structure (PDB id: 2Q37) in complex with (S)-allantoin (black spheres) 27 and Urah structure (PDB id: 2H0F) in complex with 8-azaxanthine (blue spheres) 28; orientation and colors are as in Fig 3c. (e) A 90-degrees rotation of D along the vertical axis.

ACS Chemical Biology 1

2

3

b

M

1

2

3

cb

75 63 48 35 25 20 17 -

ed

dc

6 5

* *

4 3 2 1 0

fe [θ] (deg cm2 dmol-1)

20000 15000 10000 5000 0

kcat/KM · 106 (s-1M-1)

kDa M 245 180 135 100 -

kcat/KM · 106 (s-1M-1)

aa

[θ] (deg cm2 dmol-1)

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

Page 30 of 30

0.6

0.4 0.3 0.2

*

0.1 0.0

Urah

no PEG MAL-PEG NHS-PEG

0.5

Urad

20000 15000 10000 5000 0 -5000

-5000 220

240

260

280

ACS Paragon Plus Environment 220 240 260 320 340

300

wavelength (nm)

280

300

320

340

wavelength (nm)

Figure 5. Characterization of PEG-conjugated PtAlls. (a-b) SDS-PAGE of unmodified and PEGylated PtAlls after Biosafe® Coomassie (a) and PEG-specific barium iodide dye (b) staining. Lane M, molecular weight standards; lane 1, unmodified PtAlls; lane 2, MAL-PEG PtAlls; lane 3, NHS-PEG PtAlls. (c-d) Urah (c) and Urad (d) catalytic efficiency of unmodified (black), MALPEG (red) and NHS-PEG (blue) PtAlls. *p < 0.05 versus unmodified PtAlls. (e-f) Time-evolution of circular dichroism spectra of solutions containing MAL-PEG (e) or NHS-PEG (f) in the presence of PEGylated DrUox and 200 µM urate, 20 mM potassium phosphate, pH 7.4, at 37 °C. Spectra were acquired every 2 minutes.

Diatom Allantoin Synthase Provides Structural ... - ACS Publications

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