Crystal Structures of Blasticidin S Deaminase (BSD)

The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of ∼0.6 Å, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 Å. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate. The set of blasticidin S (BS) and blasticidin S deaminase (BSD) is a widely used selectable marker for gene transfer experiments. BSD is a member of the cytidine deaminase (CDA) family; it is a zinc-dependent enzyme with three cysteines and one water molecule as zinc ligands. The crystal structures of BSD were determined in six states (i.e. native, substrate-bound, product-bound, cacodylate-bound, substrate-bound E56Q mutant, and R90K mutant). In the structures, the zinc position and coordination structures vary. The substrate-bound structure shows a large positional and geometrical shift of zinc with a double-headed electron density of the substrate that seems to be assigned to the amino and hydroxyl groups of the substrate and product, respectively. In this intermediate-like structure, the steric hindrance of the hydroxyl group pushes the zinc into the triangular plane consisting of three cysteines with a positional shift of ∼0.6 Å, and the fifth ligand water approaches the opposite direction of the substrate with a shift of 0.4 Å. Accordingly, the zinc coordination is changed from tetrahedral to trigonal bipyramidal, and its coordination distance is extended between zinc and its intermediate. The shift of zinc and the recruited water is also observed in the structure of the inactivated E56Q mutant. This novel observation is different in two-cysteine cytidine deaminase Escherichia coli CDA and might be essential for the reaction mechanism in BSD, since it is useful for the easy release of the product by charge compensation and for the structural change of the substrate. The zinc ion plays crucial roles in forming the catalytic centers of enzymes and stabilizes the three-dimensional structures of proteins (1McCall K.A. Huang C. Fierke C.A. J. Nutr. 2000; 130: 1437S-S1446SCrossref PubMed Google Scholar, 2Christianson D.W. Cox J.D. Annu. Rev. Biochem. 1999; 68: 33-57Crossref PubMed Scopus (321) Google Scholar, 3Coleman J.E. Curr. Opin. Chem. Biol. 1998; 2: 222-234Crossref PubMed Scopus (330) Google Scholar). Blasticidin S deaminase (BSD, 2The abbreviations used are:BSDblasticidin S deaminaseBSblasticidin SBsB. subtilisCDAcytidine deaminaseEcE. coliMADmultiwavelength anomalous diffractionMmM. musculusOH-BSdeaminohydroxy-BSScS. cerevisiaer.m.s.d.root mean squared deviation. EC 3.5.4.23) from Aspergillus terreus strain S-712 (ATCC 28865) is a zinc-dependent homotetrameric enzyme composed of one zinc ion and 130 amino acids per subunit (4Kimura M. Kamakura T. Tao Q.Z. Kaneko I. Yamaguchi I. Mol. Gen. Genet. 1994; 242: 121-129Crossref PubMed Scopus (90) Google Scholar, 5Kimura M. Sekido S. Isogai Y. Yamaguchi I. J. Biochem. 2000; 127: 955-963Crossref PubMed Scopus (16) Google Scholar). It catalyzes the conversion of a cytidine derivative blasticidin S (BS) into non-toxic deaminohydroxy BS (OH-BS) (Fig. 1A) (6Yamaguchi I. Shibata H. Seto H. Misato T. J. Antibiot. 1975; 28: 7-14Crossref PubMed Scopus (32) Google Scholar, 7Yamaguchi I. Seto H. Misato T. Pesticide Biochem. Physiol. 1986; 25: 54-62Crossref Scopus (8) Google Scholar). BS is a potent antifungal and cytotoxic peptidyl nucleoside antibiotic (8Takeuchi S. Hirayama K. Ueda K. Sakai H. Yonehara H. J. Antibiot. Ser. A. 1958; 11: 1-5PubMed Google Scholar). The structural determination of the 50S ribosomal subunit and BS complex revealed a mechanism inhibiting peptide translocation in protein synthesis (9Hansen J.L. Moore P.B. Steitz T.A. J. Mol. Biol. 2003; 330: 1061-1075Crossref PubMed Scopus (323) Google Scholar). Because the cytidine core of the BS molecule tightly binds to the corresponding guanine base at the P-site of the 50S ribosome with a Watson-Crick base pair, the uridination of the cytidine core by deamination prevents the induction of the interaction with guanine. Because of this property, BS and BSD are widely used as reporting markers for transformation in the genetic engineering of eukaryotic cells (10Kimura M. Takatsuki A. Yamaguchi I. Biochim. Biophys. Acta. 1994; 1219: 653-659Crossref PubMed Scopus (88) Google Scholar). blasticidin S deaminase blasticidin S B. subtilis cytidine deaminase E. coli multiwavelength anomalous diffraction M. musculus deaminohydroxy-BS S. cerevisiae root mean squared deviation. BSD is a member of the cytidine deaminase (CDA) family and has high sequence homology to tetrameric CDAs such as Bacillus subtilis CDA (BsCDA), Saccharomyces cerevisiae CDA (ScCDA), human CDA (HsCDA), and mouse CDA (MmCDA) (11Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 12Xie K. Sowden M.P. Dance G.S.C. Torelli A.T. Smith H.C. Wedekind J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8114-8119Crossref PubMed Scopus (81) Google Scholar, 13Chung S.J. Fromme J.C. Verdine G.L. J. Med. Chem. 2005; 48: 658-660Crossref PubMed Scopus (62) Google Scholar, 14Teh A-H. Kimura M. Yamamoto M. Tanaka N. Yamaguchi I. Kumasaka T. Biochemistry. 2006; 45: 7825-7833Crossref PubMed Scopus (45) Google Scholar). CDA is a zinc-dependent enzyme of the pyrimidine salvage pathway and catalyzes the reaction of cytidine to produce uridine. The proposed reaction mechanism suggests that a deprotonated water molecule is provided by the reaction between a zinc ion and a catalytic glutamate residue, and the amino group of the substrate nucleoside is replaced by a hydroxyl group. The CDA family shares the two common sequence motifs C(H)XE and PCXXC, which are involved in catalysis and zinc binding, respectively (15Reizer J. Buskirk S. Bairoch A. Reizer A. Saier Jr., M.H. Protein Sci. 1994; 3: 853-856Crossref PubMed Scopus (18) Google Scholar). In the CDA family, two different types of cytidine deaminase structure have been revealed: tetrameric CDAs, which are assembled with a 222 symmetry and their zinc ligands comprise three cysteines (CCC) (11Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar); and dimeric CDAs, which are composed of two pseudodimer subunits and take one histidine and two cysteines (HCC) as zinc ligands (16Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar). CCC coordination is rarely found in the catalytic centers of zinc enzymes, because the excessive negative charge of additional deprotonated cysteine ligands might reduce the catalytic activity of dimeric CDAs. HsCDA is pharmacologically important in the metabolism of cytidine-derived anticancer drugs (17Müller W.E.G. Zahn R.K. Cancer Res. 1979; 39: 1102-1107PubMed Google Scholar); therefore, information on the structure and function of CDAs will be valuable for the detoxification of drug-sensitive hematopoietic cells (18Steuart C.D. Burke P.J. Nat. New Biol. 1971; 223: 109-110Crossref Scopus (218) Google Scholar). Thus, information on the reaction mechanism, including the charge compensation of the zinc environment, has been analyzed and discussed (19Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. Biochemistry. 1996; 35: 1335-1341Crossref PubMed Scopus (79) Google Scholar, 20Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. Biochemistry. 1997; 36: 4768-4774Crossref PubMed Scopus (85) Google Scholar, 21Carlow D.C. Carter Jr., C.W. Mejlhede N. Neuhard J. Wolfenden R. Biochemistry. 1999; 38: 1335-1341Crossref Scopus (36) Google Scholar). The zinc ion takes various numbers and kinds of ligands because of its zero ligand-field stabilization energy owing to its having a filled d orbital and its function as a Lewis acid for accepting pairs of electrons. In particular, its coordination flexibility for polypeptide chains appears to be essential in many types of enzymatic reaction. As observed in crystallographic studies to date, the coordination geometry of the zinc ion varies and the zinc ion appears to act as a powerful electrophilic catalyst through the activation of an accompanying water molecule and a carboxylate group. The zinc ion is classified as a borderline cation according to its polarizability in hard and soft acid/base (HSAB) theory; it mostly prefers moderate nitrogen ligands although it can also accept hard and soft anions. As a result, four amino acid side chains, i.e. histidine, aspartic acid, glutamic acid, and cysteine, have been observed as protein ligands. Most reaction mechanisms of zinc enzymes have been discussed in terms of bond valence buffer theory. However, the dynamic properties of catalytic zinc remain to be elucidated, since the measurement of the properties is difficult because of the silent character of zinc in various spectroscopic methods. To elucidate the novel substrate recognition and reaction mechanisms; here we report the crystal structures of BSD in six states: native, substrate-bound, product-bound, cacodylate-bound, substrate-E56Q mutant, and R90K mutant. The appearance of two different zinc coordination structures suggests charge compensation resulting from dynamic adjustments in coordination in several crystalline states. Structural Determination of BSD—Recombinant BSD and its mutant were expressed in E. coli, purified and crystallized according to the procedure described in previous reports (5Kimura M. Sekido S. Isogai Y. Yamaguchi I. J. Biochem. 2000; 127: 955-963Crossref PubMed Scopus (16) Google Scholar, 22Nakasako M. Kimura M. Yamaguchi I. Acta Crystallogr. Sect. D. 1999; 54: 547-548Crossref Scopus (8) Google Scholar). Crystallization was carried out using a solution of 20% (w/v) PEG8000, 50 mm magnesium chloride, and 0.1 m sodium cacodylate at pH 7.0 as precipitant. The cacodylic ion occupied the active site of BSD in the crystal obtained from this solution; therefore, it was called a cacodylate-bound crystal, and the cacodylate buffer was changed to 50 mm Tris (pH 7.0) to obtain completely native crystals. These crystals belong to the space group P212121, and one asymmetric unit contains one BSD tetramer. The multiwavelength anomalous diffraction (MAD) method was used to determine phases, and the data set was collected under cryogenic conditions using the Rigaku R-AXIS IV at the BL45XU-PX station of SPring-8, Harima, Japan, as the first attempt to perform a trichromatic procedure (23Yamamoto M. Kumasaka T. Fujisawa T. Ueki T. J. Synchrotron Rad. 1998; 5: 222-225Crossref PubMed Scopus (51) Google Scholar). This procedure enables us to take diffraction images with a rapid alteration of three-wavelength x-ray beams. Hence, the dispersive differences of anomalous effects can be estimated very accurately (24Kumasaka T. Yamamoto M. Yamashita E. Moriyama H. Ueki T. Structure. 2002; 10: 1205-1210Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The data for structural refinements were also collected separately. All diffraction images were processed using an HKL suite (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38572) Google Scholar) (see Table 1). The positions of the four zinc atoms were determined using Bijvoet and dispersive anomalous Patterson maps. The initial MAD phase set was calculated using the program MLPHARE (26Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar) and further improved using DM (26Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar) by noncrystallographic symmetry averaging for the tetrameric assembly of BSD. The electron density maps allowed the unambiguous modeling of 125 residues in each subunit except for one N-terminal and four C-terminal residues. Densities corresponding to cacodylic ions as well as zinc ions were observed in the anomalous difference Fourier map using the collected data (the f″ value of arsenic atom is ∼3.5e- at an x-ray wavelength of 1.02 Å); thus, the ion models were also included. The Ramachandran plot of the refined model was sound, because all the residues except glycines and prolines were in their most favored and allowed regions (27Laskowski R.A. McArthur M.W. Moss D.S. Thronton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). All the structural models in this study were constructed using program O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. 1991; D50: 110-119Crossref Scopus (13011) Google Scholar) and refined with CNS (29Brünger A.T. et al.Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) and Refmac (26Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar).TABLE 1Crystallographic statistics for MAD phasingλ1 (remote)λ2 (peak)λ3 (inflection)Space groupP212121Cell dimensions (Å)a = 54.7, b = 69.8, c = 145.8Wavelength (Å)1.02001.28181.2822Resolution limit (Å)2.22.22.2Observations65,70976,36774,007Unique reflections23,53224,35924,252Completeness (%)80.6 (68.7)83.4 (75.2)83.0 (74.5)24.1 (10.1)22.8 (9.2)22.5 (9.4)RmergeaRmerge = ΣhklΣi |Ii(hkl) – |/ΣhklΣi Ii(hkl)3.8 (10.2)4.7 (16.2)4.8 (17.0)Phasing Resolution range (Å)20.0–2.2 Number of zinc sites4 FOMbMean figure of merit: Mlphare/DM0.49/0.90a Rmerge = ΣhklΣi |Ii(hkl) – |/ΣhklΣi Ii(hkl)b Mean figure of merit Open table in a new tab Structural Determination of Substrate and Product Complexes—All the substrate/product-bound crystals were obtained by cocrystallization in the presence of 1 mm BS/OH-BS under the same crystallization conditions in the cacodylate-bound state. OH-BS was produced from BS by an enzymatic process. A marginally active E56Q mutant and a charge-compensation residue R90K mutant were constructed by site-directed mutagenesis using a PCR technique. All the crystals except one had a similar lattice parameter with the same space group to the crystal in the native state, and their initial structures were immediately determined by rigid body refinement of the native structure. On the other hand, only the crystal of the substrate-native enzyme complex belonged to a different space group, P43212, despite the similar crystallization conditions. The crystal contained two subunits in an asymmetric unit and its structure was determined by the molecular replacement method on the basis of the structure of the substrate-unbound state using the program AMoRe (26Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar). During the structural refinement of the substrate-native complex, 2FO - FC maps showed a double-headed electron density near the catalytic zinc ion that was likely to be the reaction intermediate of BS. The corresponding molecular models of such BS states (see Figs. 1 and 2, and text) were constructed with MOLDA (30Yoshida H. Matsuura H. J. Chem. Software. 1997; 3: 147-156Crossref Google Scholar) and optimized with MOPAC7 (31Stewart J.J.P. J. Comp. Aid. Mol. Design. 1990; 4: 1-105Crossref PubMed Scopus (2816) Google Scholar). The structural restriction of the model was included except the torsion and bond angle between the pyrimidine and carbohydrate rings in the structural refinement with CNS and Refmac. Overall Structure—The tetrameric BSD structure is 45 × 45 × 40 Å (Fig. 1B). The refined BSD model consists of four identical peptides of 125 amino acids (A-, B-, C-, and D-chains). All subunits have identical folding patterns and are related by a 222 point group symmetry. In fact, the root mean square differences among the four subunits are within 0.4 Å for the main chain atoms. Each subunit is composed of a six-stranded β-sheet, sandwiched by α-helices (Fig. 1C) and is classified into the modified Greek key motif similarly to CDAs. The structure of the N-terminal-half of one monomer (1–80) begins with an α-helix (α1, Gln5-Ser20). This α-helix is embraced by an anti-parallel β-sheet (β1, Val29-Ser36; β2, Ile40-Val44; β3, Cys73-Gly78). An additional α-helix (α2, Ala55-Ala65) protrudes from the other side of the β-sheet wall. A long lariat-like region (loop β2-α2: Asn45-Pro53) connects the β-sheet (β1, β2, β3) and α2. A very short strand (β4, Ile84-Leu85) and a continuous loop connect the latter half of the monomer. This portion contains two anti-parallel strands (β5, Lys103-Lys107; β6, Thr114-Gly117) and two α-helices (α3, Cys88-Leu98; α4, Arg119-Leu121). In this domain, all secondary structures have the same orientation except α4. The crystal structures of the native and substrate-bound states (Fig. 1, B and C) are very similar, as indicated by the r.m.s. difference for all protein atoms at ca. 0.6 Å despite of the different space groups. Active Site Pockets and Substrate Recognition—The active site is located at the bottom of a deep (10 Å in depth) and narrow (5 Å in width) pocket formed by the helices α2 (Tyr47A, Cys54A, Glu56A), α3 (Cys88A, Cys91A) and residues from two adjacent subunits (Tyr126B and Phe49C) (Fig. 1C). The three aromatic residues form the side wall of the pocket, and Tyr126 from the adjacent subunit likely plays the role of a lid for the pocket entrance. Part of the pocket is composed of a loop connecting β2 and α2 in the adjacent subunit. The pocket surface is positively charged because of Arg82 and the zinc ion (Fig. 2). Furthermore, a flexible C-terminal region (Val127-Gly130) extends into the pocket. The recognition scheme with the pyrimidine ring of BS is similar to that between EcCDA and cytidine (16Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar). However, four amino acids (Ser28, Phe49, Arg82, and Tyr126) interact with the carbohydrate (enopyranuronic acid) portion and the additional tail portion including the guanidium group (Fig. 2). To expand the active site pocket for accepting the larger tail portion, BSD folds the loop regions in a more compact manner than other CDAs: (i) strand β1 is shortened, (ii) loop α1-βl protrudes to the molecular surface, and (iii) loop β2-α2 occupies the inside of the molecule. Tetramer Formation—Subunit interfaces of the tetramer are located near active site pockets. The interface is formed by the loop (residues 47–52) of β2 to α2 and four α2 helices from all subunits at the symmetric center. Tyr47, His48, Phe49, and Asp97 form a hydrogen bond network to stabilize subunit association. The mutation of these residues probably influences tetramer formation, and the H48Q mutant of BSD shows no catalytic activity (data not shown). Additionally, two interactions tightly support the tetramer: (i) α2 and the loop (residues 23–28) of α1-β1, and (ii) C-terminal residues 124–126 and two helices α3 from neighboring subunits. In the formation of the tetramer architecture, each Arg90 of four subunits may play a key role. Each arginine faces each other at the center of the tetramer interface, and shows two alternative conformations proximal and distal to the zinc (Fig. 3A). Arg90A (Arg90 of subunit A) in the proximal configuration forms hydrogen bonds with three main chain carbonyl groups: Gly51A, Thr50C, Thr50D. The latter two carbonyls also form hydrogen bonds with the adjacent Arg90B in the distal configuration. This hydrogen bond network tightly connects the subunits to each other. The vacant space of the tetrameric center is likely occupied by two water molecules. Four residual peaks that were assigned to water were observed. However, the distances between the two pairs of peaks were too short to be hydrogen bonds. Therefore, two alternate arrangements of the four arginines and two water molecules are proposed, as shown in Fig. 3A. In one arrangement (cyan atoms) Arg90A and Arg90C are in the proximal configuration and Arg90B and Arg90D are in the distal configuration. In the other arrangement (yellow atoms), Arg90A and Arg90C are in the distal configuration and Arg90B and Arg90D are in the proximal configuration. Compared with other CDAs, the pool of water molecules observed in the tetramer interfaces of BsCDA and MmCDA was eliminated in BSD (Fig. 3). This is the result of the loop β2-α2 occupation for expanding active site pocket as described before. To determine the role of Arg90, three mutants, R90K, R90A, and R90T, were constructed and expressed in E. coli. However, R90K could only be expressed as a soluble protein and purified using a BS affinity column. This result suggests that the positive charge of Arg90 is essential to enzyme function. The R90K mutant has a similar structure to the native enzyme except for the tetramer interface region. Each Lys90 employs two new water molecules, and the tetrameric center recruits a chloride ion that might compensate for the positive amino group of Lys90 (Fig. 3B). Putative Reaction Scheme—In the structure in the substrate-bound state, the substrates were unambiguously identified in the electron density map. An unexpected feature in the electron density map of BS was observed at the 4-amino (4-NH2) group of the cytidine core near the zinc ion (Fig. 4). Although the density corresponding to the amino group of the substrate or the hydroxyl group of the product was negligible, a double-headed electron density was detected around the catalyzed 4-amino group, where an amino group was exchanged for a hydroxyl group during catalytic reaction. Either of the heads could be assigned to a nitrogen or oxygen atom of unit occupancy, and the temperature factor of each atom was comparable to that of adjoining atoms in the refined model. Thus, in accordance with the suggested reaction scheme (Fig. 1A), the double-headed density could be interpreted as the amino and hydroxyl groups in the reaction intermediate of BS (BS*). However, a tear drop-shaped density was observed in the omit map in subunit A (Fig. 4C), and this density could not be satisfactorily assigned to the hydroxyl group predicted in the model. This suggests that the structure exists in a state between the enzyme-water-BS complex and the tetrahedral intermediate 1 in Fig. 1A, whereby the hydroxyl group might be in an ionic state rather than covalently bonded with the C4 atom of the pyrimidine ring. Zinc Coordination and Substrate Binding in Six States—The structure of the active center in the native state shows that the zinc ion coordinated in a tetrahedral geometry with Cys54, Cys88, and Cys91 (Fig. 5A and Table 2). This geometry is shared by other CDA structures. As proposed for the reaction mechanism of CDA (16Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar), a water molecule usually accompanying the zinc ion probably replaced the fourth ligand shown as Wat1 in Fig. 5A. This complete native structure with no substrates or inhibitors contains three water molecules, including Wat1, around the catalytic center. The positions of the water molecules correspond to those of the 4-amino group; one of the water molecules, Wat5, occupies the leaving ammonia binding site. This geometry is also observed in the OH-BS complex (Fig. 5D) and cacodylic acid binding states.TABLE 2Crystallographic statistics for refinementNativeSubstrate complex (E56Q)Substrate complexProduct complexCacodylate complexR90K mutantSpace groupP212121P212121P43212P212121P212121P212121Cell dimensions (Å)a = 54.12, b = 68.76, c = 146.43a = 55.33, b = 69.58, c = 145.99a = 54.69, c = 197.63a = 54.30, b = 69.28, c = 146.54a = 54.67, b = 69.29, c = 144.71a = 54.80, b = 69.95, c = 145.84Resolution limit (Å)1.51.81.81.51.81.6Unique reflections88,09448,59928,55089,14750,04874,811Completeness (%)99.391.598.599.894.199.933.233.632.233.933.66.3RmergeaRmerge = ΣhklΣi Ii(hkl) – /ΣhklΣi Ii(hkl)7.85.77.29.15.77.0Refinement Resolution range (Å)50.0–1.550.0–1.850.0–1.850.0–1.550.0–1.850.0–1.6 Rcryst/Rfree (%)bRcryst = Σhkl Fobs(hkl) – Fcalc(hkl)/ΣhklFobs(h kl), where Fobs and Fcalc are the observed and calculated structure amplitudes, respectively. Rfree was calculated for 5% of the reflections set aside during structure refinement17.4/19.917.6/22.120.0/22.617.3/18.517.9/21.118.5/21.4 Non hydrogen atoms4,4914,4932,0704,5174,0814,135 R.m.s.d. length (Å)/angle (°)cDetermined by Refmac (26) using Engh and Huber parameters (46)0.008/1.10.012/1.50.013/1.40.008/1.20.013/1.40.011/1.2 Average B factor (Å2)16.023.626.714.924.819.3a Rmerge = ΣhklΣi Ii(hkl) – /ΣhklΣi Ii(hkl)b Rcryst = Σhkl Fobs(hkl) – Fcalc(hkl)/ΣhklFobs(h kl), where Fobs and Fcalc are the observed and calculated structure amplitudes, respectively. Rfree was calculated for 5% of the reflections set aside during structure refinementc Determined by Refmac (26Number Collaborative Computational Project Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar) using Engh and Huber parameters (46Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2545) Google Scholar) Open table in a new tab In the substrate-bound state, the zinc coordination geometry is classified to be distorted trigonal bipyramidal rather than tetragonal (Fig. 5C). From the tetrahedral geometry, the zinc ion apparently shifts ∼0.6 Å toward the hydration water molecule hydrogen-bonded with Arg90 and enters the triangular plane of three cysteine Sγ atoms (Table 2), despite the corresponding structures of the polypeptide chains being nearly identical. At the same time, the water molecule (Wat2) exhibits a small but significant positional shift of ca. 0.4 Å. The positional shift of Wat2 is associated with the alternative conformation of Arg90, which faces the center of enzymatic tetrameric 222 symmetry. Thus, the zinc coordination shift may be influenced by the conformation of Arg90 and the signal of the shift may be transferred to other subunits through Wat3 as shown in Fig. 3A. The E56Q mutant and BS complex each show a similar conformation to the putative intermediate complex (Fig. 5B). However, this structure showed a weak electron density of the fourth water ligand, and the 4-amino group is far from zinc and interacts with Gln56Oϵ. We examined four possibilities of ambiguity of the Oϵ/Nϵ inversion and substrate/product. The two possibilities of Gln56 χ3 inversion could be distinguished by the temperature factors of Oϵ and Nϵ. From the result, we conclude that there is an interaction between the 4-amino group and GlnOϵ and that there are two possible structures: (i) the substrate BS is maintained in its 4-amino group; and (ii) the substrate is processed to an OH-BS product and the 4-amino group is substituted for a hydroxyl group. In the case (i), the 4-amino group and the heterocyclic N3 nitrogen of the cytidine core should interact with GlnOϵ and GlnNϵ, and Wat1 should not be stably coordinated to zinc in the E56Q mutant before binding to the substrate. The possibility of Wat1 existence might be small because of the result of the structure refinement even in the residual density observed in the FO - FC map. On the other hand, this structure could be considered to be trapped the exiting product in the case (ii). The 4-OH group and the N3 nitrogen would interact with GlnOϵ and GlnNϵ there, respectively. However, the interaction in latter is unfavorable. The FO - FC map also revealed the possibility of an alternative conformation of Gln56 even in low occupancy. Its χ2 angle is rotated, and the polar group is moved away from BS. Coordinate and structure factors have been deposited in the Protein Data Bank (PDB). The accession codes are PDB 1WN5, 1WN6, 2Z3G, 2Z3H, 2Z3I, and 2Z3J for the cacodylate-bound, substrate-bound, ligand free, product-bound enzymes, and E56Q and R90K mutants, respectively. Change in Coordination Geometry—The zinc coordination shift from tetragonal to trigonal bipyramidal is caused by the steric distortion of the pyrimidine ring and the carbohydrate-pyrimidine bond during substrate-product transition. The structure of the EcCDA and uridine complex suggests that the relative angle between the pyrimidine and ribose rings is distorted during the catalytic reaction (20Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. Biochemistry. 1997; 36: 4768-4774Crossref PubMed Scopus (85) Google Sch