Crystal Structure of Uroporphyrinogen III Synthase from Pseudomonas Syringae Pv. Tomato DC3000

Biochemical and Biophysical Research Communications 408 (2011) 576–581

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Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000 ⇑ Shuxia Peng a,b, Hongmei Zhang a, Yu Gao a, Xiaowei Pan a, Peng Cao a, Mei Li a, Wenrui Chang a, a National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, PR China b Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China article info abstract

Article history: Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole com- Received 11 April 2011 pounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III Available online 19 April 2011 (uro’gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5 Å resolution by the single wavelength anomalous dispersion (SAD) method. Each Keywords: psU3S molecule consists of two domains interlinked by a two-stranded antiparallel b-sheet. The confor- Uroporphyrinogen III synthase mation of psU3S is different from its homologous proteins because of the ﬂexibility of the linker between Crystal structure the two domains, which might be related to this enzyme’s catalytic properties. Based on mutation and Mutation activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Enzymatic activity Tetrapyrroles Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction believed that the inversion of the D ring is not just a simple swop of its acetate and propionate side chains, but actually involves the Natural tetrapyrroles act as cofactors for multiple enzymes rearrangement of the whole pyrrole ring [5]. The carbon atom C20 involved in important metabolic and catalytic processes within forms a covalent bond with C19 of the D ring directly to generate the cell, such as oxygen transport (heme), photosynthesis (chloro- uro’gen I without U3S. With the catalysis by U3S, the carbon atom phyll), methionine synthesis (vitamin B12), nitrite and sulfite C20 of HMB first forms a covalent bond with the carbon atom C16 assimilation (siroheme), and methane production (coenzyme of the D ring, and a spirocyclic pyrrolenine intermediate is created F430). Since these compounds are brightly colored, they are called [6,7]. Afterwards, the C ring and the D ring of this intermediate the pigments of life [1]. The first committed precursor of tetrapyr- break and then cyclize by bond formation between C19 and C15 role biosynthesis is d-aminolevulinic acid (ALA). Uroporphyrinogen to form the product, uro’gen III (Supplementary Fig. 1). However, III (uro’gen III), which is considered to be the last common precur- the details of this process and the functional residues in U3S are sor of all tetrapyrrole cofactors, is formed through three still not very clear. subsequent steps, each involving a different enzyme. U3S is the Several U3S enzymes from different species have been isolated third enzyme in the above series of reactions. It catalyzes the cycli- and purified [8–11]. U3S exists and functions in solution as a zation of linear hydroxymethylbilane (HMB) to form uro’gen III monomer. Several U3S crystal structures have been reported, such with the intermolecular rearrangement of the D ring [2]. In the ab- as human U3S [12], U3S from Thermus thermophilus (ttU3S) and its sence of U3S, linear HMB will spontaneously cyclize to form non- complex with the product uro’gen III [13], and U3S from physiological uroporphyrinogen I (uro’gen I) [3]. Mutation of U3S Shewanella amazonensis (saU3S) [14]. Although the overall struc- in the human body will reduce enzymatic activity to cause uro’gen tures of U3S enzymes are generally similar, with two domains con- I accumulation, which is associated with congenital erythropoietic nected by two b strands or two loops, structural comparison shows porphyria disease [4]. that the conformations of these U3S molecules are different [13], The catalytic mechanisms of HMB cyclization and D ring rear- and the catalytic mechanism of U3S has not been fully explained. rangement have been studied extensively. It is now generally Here we report the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 at 2.5 Å resolution. Several mutants

⇑ Corresponding author. Fax: +86 10 64889867. of psU3S were prepared to check their enzymatic activities. The E-mail address: [email protected] (W. Chang). experimental results indicate that two Arg residues are important

0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.064 S. Peng et al. / Biochemical and Biophysical Research Communications 408 (2011) 576–581 577 for enzymatic activity, and their possible functions in the catalytic 2.4. Activity assay process have been proposed. We also have discussed the catalytic process of U3S based on several existing structures and mutation Activity experiments and product measurement were done as information. previously described [12,21]. For each reaction, 10 ll PBGD (1.3 mg/ml) and 5 ll native or mutant psU3S (0.2 mg/ml) were placed in 255 ll buffer (20 mM Tris–HCl, pH 8.2, 0.1 M NaCl) and 2. Materials and methods preincubated for 2 min at 37 °C and then 30 ll of porphobilinogen (0.2 mg/ml at 37 °C) was added to start the reaction. After 3 min, 2.1. Cloning, expression and purification the reaction was stopped by addition of 60 ll of 6 M HCl, and the reagent was exposed to UV light at room temperature for 30 min The pspto_0129 gene encoding psU3S was amplified by poly- to convert the uro’gen I/III to uroporphyrin I/III. The sample was merase chain reaction (PCR) from P. syringae pv. tomato DC3000 centrifuged at 13,000g for 10 min. A 25 ll aliquot of the sample genomic DNA. The PCR genes were cloned into vector pHAT2 and was then injected onto a C18 reverse phase column (Alltech) and expressed in Escherichia coli strain BL21 (DE3) (Novagen) with an washed from the HPLC (Shimadzu) with a mobile phase 13% aceto- N-terminal 6-His-tag. Cells were harvested by centrifugation, nitrile/87% 1 M ammonium acetate pH 5.16 (v/v), to separate the resuspended with lysis buffer (50 mM Tris–HCl pH 8.0, 500 mM isomers uroporphyrin I and uroporphyrin III. The peaks were mon- NaCl) and sonicated for 15 min. The protein was purified through itored by a fluorescence detector (Shimadzu) with an excitation aNi2+ affinity column, subsequently followed by an anion ex- wavelength of 404 nm and an emission wavelength of 618 nm. change column Resource Q and a size exclusion column Superdex The areas of these peaks were compared with those arising from 200 (GE Healthcare), then concentrated to 20 mg/ml in buffer reactions using native psU3S or a non-enzymatic control (using (20 mM Hepes, pH 7.2, 20 mM NaCl) for crystallization. The seleno- 5 ll 0.2 mg/ml lysozyme instead) to estimate psU3S enzymatic methionine-substituted (SeMet) derivative was produced by activity. expression in an E. coli methionine auxotrophic strain B834 (DE3), growing in M9 minimal media supplemented with seleno- methionine. The expression and purification procedures of SeMet-psU3S and psU3S mutants were the same as that of the na- 3. Results and discussion tive protein. In order to measure enzymatic activity, the enzyme porphobi- 3.1. Overall structure of psU3S linogen deaminase (PBGD) from P. syringae pv. tomato DC3000 was cloned and expressed, using the same cloning vector, expres- The final model of the psU3S crystal structure consists of two sion vector, and expression conditions as psU3S. The PBGD protein monomers (A and B) per asymmetric unit. In common with other was purified through a Ni2+ affinity column, followed by size exclu- U3S structures, it belongs to the HemD-like fold family [22]. Each sion chromatography with Superdex 200 (GE Healthcare). The pro- psU3S molecule is composed of two domains. Domain 1 includes tein was concentrated to 1.3 mg/ml for activity measurement. residues 2–32 and 169–258, with a five-stranded parallel b-sheet (b1, b2, b10–b12) surrounded by six a-helices (a1, a8–a12). Domain 2 comprises residues 40–162, with a five-stranded parallel 2.2. Crystallization and data collection b-sheet (b4-b8) surrounded by six a-helices (a2–a7). The two domains are connected by a two-stranded antiparallel b-sheet, The psU3S crystals were grown by the sitting drop vapor diffu- including residues 33–39 (b3) and residues 163–168 (b9) sion method. A volume of 1 ll protein solution was mixed with an (Fig. 1A). Although the topologies of the two domains are similar, equal volume of reservoir solution containing 20% PEG 3350, 0.2 M their structures cannot be overlapped completely. Superposition tri-Na citrate, equilibrating against 100 ll reservoir solution. Crys- of the two domains (121/123 Ca) with the program DaliLite [23] tals appeared in clusters after growing for about a month at 8 °C yields a root-mean-square-deviation (RMSD) of 3.1 Å. and, finally, good single crystals were obtained by the microsee- All the residues except the first Met have been traced in the ding method. The crystals were dipped into a cryoprotectant of psU3S structure. Because of the invisible electron density, residues mixed oil (paraffin oil and NVH oil at a ratio of 7:3), and flash- 108–114 of molecule A were not built successfully, while in mole- cooled through a nitrogen-gas stream at 100 K for data collection. cule B they are represented as a flexible loop (Fig. 1B), which is lo- Diffraction data of both native psU3S and SeMet-psU3S were col- cated on the surface of domain 2 and near the opening of the lected at BL17U of Shanghai Synchrotron Radiation Facility. The pocket between the two domains. Structure comparison of mole- psU3S crystal belongs to space group P21. All data were processed cule A and molecule B yields an RMSD of 0.59 Å [23]. The two and scaled with HKL-2000 [15]. monomer molecules in the psU3S structure interact with one other by hydrogen bonds between residues Arg219 in one molecule and 2.3. Structure determination and refinement Glu147/Glu151 in the other, and residues Glu143 in molecule A with Glu147 in molecule B (Fig. 1D). The structure of psU3S was solved by the single wavelength anomalous dispersion (SAD) method, with four selenium atoms per asymmetric unit. The initial phase was obtained by the pro- 3.2. Comparison with other U3S structures gram Autosol in the PHENIX software suite [16], and further im- proved by density modification with RESOLVE [17]. The program PsU3S shares 26% sequence identity with human U3S. The Autobuild was used to build 80% of the main chain of the whole RMSD between the overall structure of psU3S and that of human structure. The additional residues were rebuilt manually by COOT U3S (PDB ID:1JR2) is 4.7 Å [23]. RMSDs on structurally equivalent [18], and the model was refined with the native diffraction data residues of domain 1 (121/123 Ca) and domain 2 (122/124 Ca) in CNS [19]. The stereochemical quality of the refined structure of the two molecules are both 2.5 Å. Although the overall struc- was checked with the program PROCHECK [20]. A summary of data tures of the two enzymes are similar, there is an obvious difference collection and structure refinement statistics is listed in Supple- between their conformations. Superposition of Ca atoms of the mentary Table 1. Figs. 1–3 were prepared with the program Pymol two structures on domain 1 shows that their domains 2 are twisted (DeLano Scientific, LLC). with an angle of about 30o [24] (Fig. 2A). 578 S. Peng et al. / Biochemical and Biophysical Research Communications 408 (2011) 576–581

Fig. 1. (A) Ribbon representation of psU3S monomer with different structural elements labeled. (B) Superposition of molecule A (pink) with molecule B (cyan) in psU3S. Residues 108–114 in molecule B are represented as a loop, which is not built in molecule A. (C) Overall structure of psU3S in an asymmetric unit. Molecule A is shown in pink and molecule B in cyan. The interactions between the two molecules are shown in the red rectangle. (D) Molecule A (pink) and molecule B (cyan) interact with each other via hydrogen bonds (red dashed lines). The residues forming hydrogen bonds are shown as sticks. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Comparing the overall structure of psU3S with that of ttU3S- catalytic pocket between the two domains may adopt open, closed, uro’gen III complex (PDB ID: 3D8N) displays an RMSD of 4.7 Å or any intermediate state, since the flexible linker leads to the [23]. The sequence identity of the two enzymes is only 16%. Over- random relative motion of the two domains in the solution lapping Ca atoms of domains 1 of psU3S and ttU3S, shows a environment. displacement of domains 2 with a rotation angle of about 20° (Fig. 2B) [24]. In the structure of ttU3S-uro’gen III complex, the 3.3. The potential active sites of psU3S connection of the two domains is much more flexible, with a less regular conformation of two loops, whereas in psU3S and human It has been proposed that the active sites for binding substrate U3S structures the linkers are two antiparallel b strands. The and catalysis lie in the pocket area [12,13]. According to the charge rotation and bending of the two domains in U3S are related to distribution of human U3S, psU3S and ttU3S surfaces (Fig. 2C–E), the flexibility of the two fragments between them. most of the enzyme’s surface is negatively charged, whereas the Superposition of human U3S, psU3S and ttU3S-uro’gen III com- pocket region between the two domains is mainly positively plex shows that their active pockets are in different open states charged, and is able to bind the negatively charged substrate. More- (Fig. 2C–E). The opening of the pocket between the two domains over, the product in the ttU3S-uro’gen III complex structure is held of human U3S is the largest one, and adopts the most open confor- into the pocket region between the two domains [13]. Nuclear mag- mation. The opening of the ttU3S pocket is the smallest, and it netic resonance perturbation studies revealed that some highly adopts a relatively closed conformation. The structure of the psU3S conserved residues lining the pocket surface of U3S might partici- pocket lies between these two and closer to that of ttU3S. It was pate in catalytic activity in some eukaryotes [25]. A sequence align- proposed in the previous study that the more open conformation ment of U3S from different species [26] showed that some of these might facilitate substrate binding and product release, while the active residues were also conserved in psU3S (Supplementary closed state might be required for cyclization of the HMB rings Fig. 2), such as Leu35, Ser63, Gly90, Thr93, Arg142, Tyr162, [12]. The psU3S structure we determined is more similar to the Ser190, Gly193, Leu243 (Fig. 3A). Tyr162 was confirmed as playing closed conformation, but it does not bind any substrate or product. an important role in enzymatic activity, since the mutation of this Therefore, it is proposed that in the absence of substrate or Tyr residue in U3S from both human and Anacystis nidulans resulted product, U3S may take a variety of conformation states and the in a significant decrease in activity [12,21]. Moreover, the Thr228A- S. Peng et al. / Biochemical and Biophysical Research Communications 408 (2011) 576–581 579

Fig. 2. (A) Structural comparison of psU3S (cyan) with human U3S (PDB ID: 1JR2) (magenta) by superposition of domain 1. (B) Structure comparison of psU3S (cyan) with ttU3S-uro’gen III complex (PDB ID: 3D8N) (yellow) by superposition of domain 1. The product uro’gen III is shown as gray sticks. Electrostatic surface representations of human U3S (C), psU3S (D) and ttU3S (E) are observed in different U3S structures. Red represents a negative charge, and blue represents a positive charge. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

la mutant in human U3S only retained 32% of the wild-type activity Arg219 is on the a11 helix of domain 1 near the molecular [12], but this site was replaced by Val220 in psU3S. The sequence surface, with its side chain toward the pocket region. Superpos- alignment showed its adjacent residue Arg219 was also conserved ing the overall structure of psU3S and that of ttU3S-uro’gen III in many species (Supplementary Fig. 2). complex, we found that the distance of Arg219 and product uro’- Based on the crystal structure and sequence alignment, five gen III was beyond 12 Å and there was no direct interaction be- other highly conserved residues were proposed to be involved in tween them. Then, how does Arg219 affect the catalytic process? the potential active sites and to participate in enzyme catalysis. By analysis of the structure, it was found that the side chain of They are all located on the surface of the pocket area or close to this Arg219 is close to several residues in domain 2 (5.9–7.1 Å), such region, including Glu38 and Arg164 in the linker of the two major as Gly141, Arg142, Glu143 and Leu144 (Fig. 3C). Since Arg219 is domains, Glu114 and Leu116 in domain 2, and Arg219 in domain located at the opening of the pocket between the two domains, 1(Fig. 3B). We prepared the mutants of Glu38Ala, Glu114Ala, its distance to domain 2 is so close that the substrate cannot Leu116Ala, Arg164Ala and Arg219Ala, and measured the enzy- bind into the pocket region directly; it needs a conformational matic activity of mutant and wild-type psU3S. change of psU3S. The conformational change during the sub- The results of mutation and enzymatic activity measurement strate binding process may be due to the electrostatic interaction showed that the activities of Glu38Ala, Glu114Ala and Leu116Ala between the positively charged Arg219 side chain and the nega- mutants were not changed very much compared to that of wild- tively charged carboxylate side chains of substrate HMB, and type protein, the activity of Arg164Ala mutant was reduced to hydrogen bonds would be formed between Arg219 and the sub- 70%, and mutant Arg219Ala exhibited the greatest decrease in strate. After Arg219 binding with the substrate, the conformation activity, at only 25% of the wild type activity (Fig. 4). After analyzing of psU3S would change and Arg219 would be pushed away, then the structure of psU3S, we found that Glu38 is on the interface be- the substrate could enter into the pocket area. If Arg219 was tween the two domains, but its side chain is toward the outer side mutated to Ala, the charge-charge interaction would be lost in of the pocket, therefore it may not be involved in catalytic process. this position, and the enzymatic activity decreased. On the other Glu114 and Leu116, connecting with a loop near the opening of the hand, given into that the mutation of this residue caused a sig- pocket, are a little distant from the central area of the pocket, and nificant reduction in enzymatic activity, we presume that this they may not participate in the catalytic reaction directly or may residue might be not only concerned with substrate binding, only bind substrate with weak interactions, so that their activities but also with the conformational change of U3S after binding, were generally unchanged after mutation. However, the side chains including the production of intermediate product and the flip- of Arg219 and Arg164 are directly extended into the pocket region ping of the D ring. In addition, it is noteworthy that, although (Fig. 3B), just in the core of the substrate binding and catalytic areas, the sequence alignment shows that it is Thr227 in human U3S so it is highly possible that Arg219 and Arg164 are involved in the on this corresponding site (Supplementary Fig. 2), a nearby res- catalytic process. idue, Arg230, which is also located on the surface of human U3S 580 S. Peng et al. / Biochemical and Biophysical Research Communications 408 (2011) 576–581

Fig. 3. (A) The conserved residues in psU3S that correspond to the active sites in human U3S are shown as sticks. (B) Five residues of psU3S mutated in this study are shown as sticks. (C) The residues in domain 2 that are close to Arg219 are shown as sticks, and their distances are labeled in red dashed lines. The yellow sticks represent the product uro’gen III by superposing psU3S (cyan) with ttU3S-uro’gen III complex (yellow). (D) Superposition of psU3S (cyan) and ttU3S-uro’gen III complex (yellow) showed that Arg164 could form hydrogen bonds with A ring acetate and propionate of uro’gen III. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

could form hydrogen bonds with the A ring acetate and propionate, respectively (Fig. 3D). Therefore, we infer that mutation of Arg164 side chain may lead to some loss of hydrogen bond interactions and the substrate cannot bind with the enzyme strongly, resulting in a loss of enzymatic activity. Moreover, Arg164 is a residue with a positive charge which may also help to attract the entry of the negatively charged substrate, as with Arg219. Another significant active site is Tyr162. In human U3S, its corresponding residue Tyr155 might approach the bridge carbon atom C20 and form a hydrogen bond with the hydroxyl of HMB, and this Tyr might contribute to the loss of substrate hydroxyl and formation of the azafulvene intermediate [13]. Tyr162 might take the same role in psU3S. These findings, combined with our experiments, lead to the following speculations on the catalytic mechanism of U3S. In the structure of psU3S, the D ring acetate Fig. 4. The activity analysis of psU3S mutants. The activity of wild-type psU3S was or propionate of linear HMB with a negative charge is initially considered as 100, and the activities of mutant proteins were expressed as a attracted by the positively charged polar pocket formed by percentage of wild-type control activity. residues such as Arg219 at the opening of this pocket, and forms hydrogen bond interactions with Arg219. At the same time, the conformation of psU3S changes, and the residues near the opening domain 1 and close to Arg219 in psU3S by structure superposi- of the pocket, such as Arg219, would be pushed away by the sub- tion, may play the same role as Arg219. strate to enlarge the pocket between the two domains, and the Arg164 is located at the interface of the two domains, and its other rings of HMB can enter into the pocket area. Subsequently, side chain is also extended into the pocket area. By sequence align- one side of the linear HMB keeps the interaction with Arg219, ment and structure comparison, its corresponding residue is the A ring on the other side of the linear HMB forms hydrogen His165 in ttU3S (Supplementary Fig. 2). In the ttU3S-uro’gen III bonds with residues like Arg164, and the hydroxyl connecting to complex structure, the side chain of His165 forms a hydrogen bond C20 would form a hydrogen bond with Tyr162. The large distance with the uro’gen III A ring acetate [13], and Arg164 in psU3S might between Arg219 and Tyr162 (about 15 Å in the psU3S structure) also form a hydrogen bond with the substrate or product to stabi- would avoid the C20 of HMB connecting with C19 of D ring on lize their binding in this pocket area. We superposed the psU3S the other side directly to form uro’gen I. Afterwards, the enzyme structure with the ttU3S-uro’gen III complex structure [24] and conformation would continue to change, the linker of the two found that Arg164 was very close to the product uro’gen III and domains would be bent and distorted, and the pocket of the two S. Peng et al. / Biochemical and Biophysical Research Communications 408 (2011) 576–581 581 domains would tend to close. The distance between Arg219 and [9] G.J. Hart, A.R. Battersby, Purification and properties of uroporphyrinogen III Tyr162 would decrease and the C20 of HMB would combine with synthase (co-synthetase) from Euglena gracilis, Biochem. J. 232 (1985) 151–160. C16 on the D ring selectively via loss of hydroxyl, with the syner- [10] S.F. Tsai, D.F. Bishop, R.J. Desnick, Purification and properties of gistic help of other residues, to form the spirocyclic intermediate uroporphyrinogen III synthase from human erythrocytes, J. Biol. Chem. 262 (Supplementary Fig. 3). 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