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Crystal Structure of Bacillus subtilis Guanine Deaminase

2004; Elsevier BV; Volume: 279; Issue: 34 Linguagem: Inglês

10.1074/jbc.m405304200

1083-351X

Shwu‐Huey Liaw, Yu-Jui Chang, Cheng‐Tsung Lai, Hui-Chuan Chang, Gu‐Gang Chang,

Virus-based gene therapy research

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-Å resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 Å2 per monomer. The essential zinc ion is ligated by a water molecule together with His53, Cys83, and Cys86. A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp114 and Tyr156′. The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases. Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-Å resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 Å2 per monomer. The essential zinc ion is ligated by a water molecule together with His53, Cys83, and Cys86. A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp114 and Tyr156′. The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases. Purine/pyrimidine bases and nucleotides serve as nitrogen and carbon sources and also take part in nucleotide synthesis. A deamination step is the first and the commitment step in the degradation and salvage pathways. Therefore, purine/pyrimidine deaminases play key roles in the nucleotide metabolism and have become important possibilities for anticancer and antibacterial therapy. Guanine deaminase (GD 1The abbreviations used are: GD, guanine deaminase; bGD, Bacillus subtilis GD; yCD, yeast cytosine deaminase; CDA, cytidine deaminase; eCDA, Escherichia coli CDA; bCDA, B. subtilis CD; AICAR, 5-aminoimidazole-4-carboxamide-ribonucleotide; DHX, 1, 2-dihydroxanthine; dCMPD, dCMP deaminase; RibG, riboflavin biosynthesis protein.1The abbreviations used are: GD, guanine deaminase; bGD, Bacillus subtilis GD; yCD, yeast cytosine deaminase; CDA, cytidine deaminase; eCDA, Escherichia coli CDA; bCDA, B. subtilis CD; AICAR, 5-aminoimidazole-4-carboxamide-ribonucleotide; DHX, 1, 2-dihydroxanthine; dCMPD, dCMP deaminase; RibG, riboflavin biosynthesis protein.; EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine into xanthine and ammonia, thereby irreversibly removing the guanine base from the pool of guanine-containing metabolites. Our sequence analysis suggests that two types of GDs have evolved separately. Plant, Caenorhabditis, Archaea, and some bacterial GDs belong to the cytidine deaminase (CDA) superfamily (1Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (336) Google Scholar, 2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). For example, the Bacillus subtilis GD (bGD) has been shown to be inducible with purines as nitrogen sources (3Fassbinder F. Kist M. Bereswill S. FEMS Microbiol. Lett. 2000; 191: 191-197Crossref PubMed Google Scholar, 4Nygaard P. Bested S.M. Andersen K.A.K. Saxild H.H. Microbiology. 2000; 146: 3061-3069Crossref PubMed Scopus (23) Google Scholar). On the other hand, mammalian, insect, fungal, and some bacterial GDs belong to the TIM barrel metallohydrolase superfamily (5Holm L. Sander C. Proteins. 1997; 28: 72-82Crossref PubMed Scopus (417) Google Scholar, 6Lai W.L. Chou L.Y. Ting C.Y. Kirby R. Tsai Y.C. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2004; 279: 13962-13967Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). GD is the only identified enzyme in mammals that can directly deaminate a base, and its diagnostic usefulness has been well documented, for example, in the detection of hepatoma and the identification of donor blood infected with the hepatitis C virus (7Matsunaga H. Honda H. Kubo K. Sannomiya K. Cui X. Toyota Y. Mori T. Muguruma N. Okahisa T. Okamura S. Shimizu I. Ito S.J. J. Med. Investig. 2003; 50: 64-71PubMed Google Scholar, 8Roberts E.L. Newton R.P. Anal. Biochem. 2004; 324: 250-257Crossref PubMed Scopus (9) Google Scholar). The expression of mammalian GDs has been shown to be tissue-specific and to fluctuate during development, and various GD activities have been found in cancerous breast and kidney tissues (9Yuan G. Bin B.C. McKay D.J. Snyder F.F. J. Biol. Chem. 1999; 274: 8175-8180Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Paletzki R.F. Neuroscience. 2002; 109: 15-26Crossref PubMed Scopus (33) Google Scholar, 11Canbolet O. Durak I. Cetin R. Kavutcu M. Demirici S. Ozturk S. Breast Cancer Res. Treat. 1996; 37: 189-193Crossref PubMed Scopus (58) Google Scholar, 12Durak I. Beduk Y. Kavutcu M. Suzer O. Yaman O. Oztura H.S. Canbolat O. Ulutepe S. Cancer Investig. 1997; 15: 212-216Crossref PubMed Scopus (26) Google Scholar). Different human GD isoforms have been identified and shown to modify neurotransmitter receptors at the synaptic sites during neuronal development (13Kuwahara H. Araki N. Makino K. Masuko N. Honda S. Kaibuchi K. Fukunaga K. Miyamoto E. Ogawa M. Saya H. J. Biol. Chem. 1999; 274: 32204-32214Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Thus, GD may have the potential to be an attractive candidate for the study of applications, including drug design and diagnostics.In addition to GDs mentioned above, the purine/pyrimidine deaminases in the CDA superfamily include CDAs, fungal cytosine deaminases, dCMP deaminases (dCMPDs), riboflavin biosynthesis proteins (RibGs), and RNA editing enzymes containing either an adenosine deaminase or a cytidine deaminase domain (1Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (336) Google Scholar, 2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Weiner K.X.B. Weiner R.S. Maley F. Maley G. J. Biol. Chem. 1993; 268: 12983-12989Abstract Full Text PDF PubMed Google Scholar, 15Schultz A. Nygaard P. Saxild H.H. J. Bacteriol. 2001; 183: 3293-3302Crossref PubMed Scopus (98) Google Scholar, 16Wedekind J.E. Dance G.S. Sowden M.P. Smith H.C. Trends Genet. 2003; 19: 207-216Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 17Keegan L.P. Leroy A. Sproul D. O'Connell M.A. Genome Biol. 2004; 5: 209-218Crossref PubMed Scopus (114) Google Scholar). These deaminases catalyze the zinc-assisted conversion of the amino group of the cytosine, guanine, or adenine moiety into a keto group. The substrates in the CDA superfamily are made up of similar building blocks, namely base, ribose, and phosphate (Fig. 1). A major challenge is to understand how nature has evolved the CDA fold of these various deaminases to act on their substrates. Two questions may be posed. First, are similar or dissimilar residues used to interact with the common moieties of the substrates? Second, can the specific substrate-interacting residues for each deaminase be predicted through combinations of structural and bioinformatics methods? To gain structural insights into the substrate specificity and evolution of bGD, we have solved the enzyme structure at 1.17-Å resolution.EXPERIMENTAL PROCEDURESThe protein crystallization, diffraction data collection, phase determination using selenium multiwavelength anomalous detection, and the building of an initial dimeric model have been described in our preliminary report (18Chang Y.J. Huang C.H. Hu C.H. Liaw S.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1152-1154Crossref PubMed Scopus (6) Google Scholar). The structure then underwent straightforward refinement against data to 1.17-Å resolution using CNS (19Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar). Statistics for the refined model are shown in Table I. More than 92% of the residues are in the most favored regions of Ramachandran plot, with the remaining ones located in the additional allowed regions. The side chains of Ser47, Leu76, and Thr138 have two alternative conformations, and this fact was taken into account during refinement. Figs. 2, 3B, 4A, and 4B were generated by MolScript (20Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (21Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar), Fig. 3A by BobScript (22Esnouf R.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 938-940Crossref PubMed Scopus (849) Google Scholar), and Fig. 3C by Grasp (23Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5313) Google Scholar).Table IStatistics for data collection and structural refinementData collectionSpace groupC2221Unit cell (Å)81.57, 91.27, 80.48Resolution range (Å)50-1.17 (1.19-1.17)aValues in parentheses are for the highest resolution shell.Total observations1,083,056 (13,302)aValues in parentheses are for the highest resolution shell.Unique reflections96,780 (3695)aValues in parentheses are for the highest resolution shell.Completeness (%)95.7 (70.3)aValues in parentheses are for the highest resolution shell.I/σ〈I〉37.4 (4.3)aValues in parentheses are for the highest resolution shell.Rmerge (%)5 (22.9)aValues in parentheses are for the highest resolution shell.RefinementResolution range (Å)50-1.17 (1.18-1.17)aValues in parentheses are for the highest resolution shell.Reflections (F > 0 σF)96,780 (3695)aValues in parentheses are for the highest resolution shell.Rcryst (%) for 90% data16.1 (24.6)aValues in parentheses are for the highest resolution shell.Rfree (%) for 10% data17.7 (25.4)aValues in parentheses are for the highest resolution shell.Estimated coordinate error by Luzzati plot (Å)0.11Estimated coordinate error by SigmaA plot (Å)0.13Root mean square deviationsBond lengths (Å)0.013Bond angles (°)1.6Average B-factors (Å2)Main-chain atoms (1252)9.4Side-chain atoms (1168)12.9Water molecules (450)13.5Inhibitor atoms (10)12Zinc ions (2)6.8a Values in parentheses are for the highest resolution shell. Open table in a new tab Fig. 2Structure of bGD. Ribbon views of the monomer (A) and dimer (B). The tightly bound zinc ion is shown as a sphere with its ligands, the general base glutamate, and the transition-state intermediate analogue (DHX) as ball-and-stick representations. The protein is a three-layered α/β/α structure with a central β-sheet sandwiched on either side by α-helices. The dimer is made up of one monomer colored in red, and the other colored in green (Fig. 2B was created with a similar orientation to Fig. 3C). Single letter amino acid abbreviations are used with position numbers in panel A.View Large Image Figure ViewerDownload (PPT)Fig. 3The active site of bGD.A, the 2Fo – Fc electron density map of the active site contoured at a 3 σ level and shown in cyan, and the difference anomalous map for the zinc ion contoured at a 20 σ level and shown in purple. The densities of the bound imidazole and the three water molecules (W1–W3) are highlighted in green. The active site residues and the imidazole are shown as ball-and-stick representations, and the modeled inhibitor (DHX) as magenta sticks. The zinc ion and the water molecules as magenta and red spheres, respectively. B, stereo view of the interaction networks in the active site. There are nine direct hydrogen bonds between the protein molecule and the inhibitor (see “Results and Discussion” for a detailed explanation). C, molecular surfaces of one bGD subunit are colored for electrostatic potential from –10 kBT (red) to 10 kBT (blue), whereas the surfaces of the other subunit are displayed explicitly as worms. The zinc ion is embedded at the deepest part, whereas the inhibitor lies near the cavity opening (Fig. 3C was created with a similar orientation to Fig. 2B). Single letter amino acid abbreviations are used with position numbers.View Large Image Figure ViewerDownload (PPT)Fig. 4Structural conservation and divergence of the CDA superfamily.A, stereo view of structural superposition of bGD (Protein Data Bank code 1WKQ; residues 2–110 are shown in red, and residues 130′–156′ are shown in magenta), yCD (Protein Data Bank code 1UAQ; green), and bCDA (Protein Data Bank code 1JTK; blue). These three deaminases contain the conserved strands β1–β4 and helices αA–αC, and the diverse C-terminal segments are labeled in the same color for each protein. B, superposition of the active sites of bGD (magenta), yCD (green), and bCDA (cyan). The residue numbering is labeled in the same color for each protein. C, multiple sequence alignment of bGD, yCD, bCDA, the catalytic domain of eCDA, human dCMPD (hdCMPD), the deaminase domain of B. subtilis RibG (bRibGD), and subdomains 2 and 4 of the chicken AICAR transformylase domain (AICAR2 and AICAR4). The secondary structure elements for each enzyme, derived from the crystal structures or predicted by PSI-PRED, are boxed, and those for bGD are labeled (s.s). Residues conserved in the CDA superfamily and involved in deamination are shaded in cyan, whereas the residues for the conserved hydrophobic core are shaded in yellow. In addition, the residues conserved in each member and involved in the substrate binding are shaded in red, whereas the residues that form hydrogen bonds with protein atoms for structural integrity are shaded in blue. The conserved glycine and proline residues in each family are shaded in gray. The large insertion (residues 470–532, subdomain 3) in AICAR4 is indicated by ***, is not involved in the catalytic activity, and is present in the eukaryotic enzymes only. Single letter amino acid abbreviations are used with position numbers.View Large Image Figure ViewerDownload (PPT)To identify the putative substrate recognition residues for some members, a comparative analysis of the available crystal structures in the CDA superfamily was first carried out, and then sequence similarity searches were conducted by PSI-BLAST (24Schäffer A.A. Aravind L. Madden T.L. Shavirin S. Spouge J.L. Wolf Y.I. Koonin E.V. Altschul S.F. Nucleic Acids Res. 2001; 29: 2995-3005Crossref Scopus (1112) Google Scholar). Multiple sequence alignments of 25 and 32 homologous sequences for dCMPD and RibG, respectively, were performed by ClustalW (25Chenna R. Sugawara H. Koike T. Lopez R. Gibson T.J. Higgins D.G. Thompson J.D. Nucleic Acids Res. 2003; 31: 3497-3500Crossref PubMed Scopus (4014) Google Scholar). This was followed by manual editing according to the structural information and secondary structure prediction using PSI-PRED (26McGuffin L.J. Bryson K. Jones D.T. Bioinformatics. 2000; 16: 404-405Crossref PubMed Scopus (2665) Google Scholar). Finally, the conserved residues in each family were mapped on the known structures to reveal whether they are potentially localized nearby the active site cavity.RESULTS AND DISCUSSIONThe Overall Structure—Analytical ultracentrifugation experiments demonstrated that the enzyme exists in solution as a homodimer. In the crystal there is one dimer per asymmetric unit, and there are no significant differences between the two subunits except for the N-terminal seven residues and the side chains of several arginines and lysines (root mean square deviation of 0.82 Å for all protein atoms of residues 8–156). The current model contains two extra residues (His (–2) and Ala (–1)), residues 1–156 from subunit A, and residues 2′–156′ from subunit B.Unexpectedly, bGD forms an intertwined dimer through C-terminal domain swapping (Fig. 2). The protein structure consists of a central five-stranded β-sheet (β1–β4 and β5′ of the adjacent subunit) with the strand order 2, 1, 3, 4, and 5′ and with β1 running antiparallel to other strands. The β-sheet is sandwiched by helices αA, αD1, and αE′ on one side and helices αB, αC, and αD2 on the other side. The helices αD1 and αD2 extend away from the subunit core and wrap around the adjacent subunit, leading to the C-terminal segment, residues 123–156, which is swapped. Consequently, the β5 strand of one chain forms hydrogen bonds to the β4 strand of the other in a parallel manner, thereby completing the five-stranded β-sheet of the CDA fold. The swapping results in a very extensive intersubunit interface that buries 3900 Å2 of the 10200-Å2 molecular surface area per monomer.More than 45 residues from each subunit, including many conserved residues in the GD family, are involved in dimer formation. The dimer is stabilized mainly by the formation of the wide interhelical hydrophobic packing of helices αA and αD1 with αE′ and the packing of αB, αC, and αD2 with αB′, αC′, and αD2′. In addition, there are 26 direct hydrogen bonds between the protein atoms, including salt bridges between Lys8 and Glu139, Asp49 and Arg60, and Arg94 and Asp113. Interestingly, the side chains of the conserved Arg60 from both subunits stack very well, with a distance of 3.6 Å between the guanidino groups.The Active Site Architecture and Substrate Binding—The active site contains one tightly bound metal ion, even though no metal ions were added either during protein purification or crystallization. The metal ion was identified by a very strong spherical electron density in the Fourier map and was assigned as zinc based on zinc anomalous data (18Chang Y.J. Huang C.H. Hu C.H. Liaw S.H. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1152-1154Crossref PubMed Scopus (6) Google Scholar). The zinc ion is tetrahedrally coordinated by His53 Nδ1 (2.06 Å), Cys83 Sγ (2.34 Å), Cys86 Sγ (2.28 Å), and a water molecule, WAT1 O (2.03 Å) (Fig. 3A).There was a significant electron density peak for a five-atom aromatic ring in the 2Fo – Fc and Fo – Fc electron density maps (Fig. 3A). The signal was assigned as an imidazole from elution off the nickel column, because it occupies the putative position of the imidazole group of the substrate guanine with extensive interactions (Fig. 3). The N1 atom interacts with Asn42 Nδ2 (3.29 Å) and Tyr156′ Oη (2.90 Å), and the N3 atom contacts with Asp114 Oδ1 (2.86 Å). The imidazole ring of His53 and the phenyl ring of Phe26 stack on the imidazole ring with an interplanar distance of 3.3–3.4 Å. The C2 atom also makes close contacts with Trp92′ Cη2 (3.63 Å), Phe112 Cδ1 (3.89 Å), and Tyr156′ Cϵ1 (3.52 Å). Three strong peaks in the active site were assigned as water molecules (Fig. 3A). WAT1 ligates the zinc ion as the fourth ligand. It also makes close contact with Glu55 Oϵ2 (2.58 Å) and Cys83 N (2.95 Å) and would occupy the OH2 position of the reaction intermediate. WAT2 forms hydrogen bonds with Asn42 Nδ2 (2.95 Å) and Ala54 N (3.12 Å) and also binds at the O6 position of the intermediate. WAT3 interacts with Glu81 O (2.50 and 2.88 Å in the subunits A and B, respectively) and Asp114 Oδ2 (3.09 and 3.03 Å, respectively) and would seem to be near to the NH 22 group of the intermediate or the leaving ammonium (27Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. Biochemistry. 1997; 36: 4768-4774Crossref PubMed Scopus (82) Google Scholar).On the basis of the tightly bound WAT1, WAT2, and imidazole molecules described above, the transition state analogue 1, 2-dihydroxanthine (DHX) was modeled into the active site (Figs. 1 and 3). Hypoxanthine is expected to be converted by bGD into DHX, because similar inhibitors have been used in previous crystallographic studies of Escherichia coli CDA (eCDA) and yeast cytosine deaminase (yCD) (1Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (336) Google Scholar, 2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). After modeling, energy minimization was performed by CNS as a structural refinement. The nucleophilic OH2 group of the purine ring coordinates to the catalytic zinc ion (2.05 Å) and interacts with Glu55 Oϵ2 (2.58 Å) and Cys83 N (2.98 Å). Additionally, the N1 atom makes close contact with Glu55 Oϵ1 (2.73 Å), the N3 atom with Asp114 Oδ2 (2.84 Å), the O6 atom with Ala54 N (3.11 Å) and Asn42 Nδ2 (2.96 Å), the N7 atom with Asn42 Nδ2 (3.29 Å) and Tyr156′ Oη (2.87 Å), and the N9 atom contacts with Asp114 Oδ1 (2.82 Å). In addition to the extensive hydrogen bond network, the imidazole ring of His53 and the phenyl ring of Phe26 stack on the purine ring with an interplanar distance of 3.3–3.4 Å. Also, the C8 atom of the purine ring makes close contacts with Trp92′ Cη2 (3.65 Å), Phe112 Cδ1 (3.84 Å), and Tyr156′ Cϵ1 (3.50 Å). The hydrogen bonds between the substrate and the conserved residues are virtually identical to those found in yCD and CDAs (1Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (336) Google Scholar, 2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (87) Google Scholar).Structural Conservation in the CDA Superfamily—A structural similarity search by DALI (29Dietmann S. Park J. Notredame C. Heger A. Lappe M. Holm L. Nucleic Acids Res. 2001; 29: 55-57Crossref PubMed Scopus (147) Google Scholar) reveals that bGD displays significant structural similarity to yCD, B. subtilis CDA (bCDA), eCDA, and subdomain 2 of the chicken AICAR transformylase domain, with Z-scores of 14.2, 9.9, 8.9, and 8.1, respectively (1Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (336) Google Scholar, 2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (87) Google Scholar, 30Wolan D.W. Greasly S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (36) Google Scholar). Two classes of CDAs have been identified: (i) a dimeric and pseudo-tetrameric form such as eCDA that utilizes one histidine and two cysteines for zinc ion coordination; and (ii) a tetrameric form such as bCDA that uses three cysteines instead. Detailed structural comparisons reveal that the conserved structural elements in the CDA fold include the strands β1–β5 and the helices αA–αC (Fig. 4A). The main chain atoms of the 65–70 structurally equivalent residues overlay with a root mean square deviation of 1–1.35 Å and 8–24% sequence identity. The strong conservation of the tertiary structures of these domains suggests that they are evolutionarily descended from a common structural fold, the CDA fold. To date, enzymes of this CDA fold exist as an oligomer and lack disulfide bonds. Similarly, the active site cavity in the CDA superfamily is mainly made up of the αA-β1, β2-αB, β3-αC, and β4-αD loops and the C-terminal tails.The dimeric yCD has an intramolecular active site (2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), whereas the dimeric bGD contains an intersubunit one. In contrast, the active site of the tetrameric bCDA is built from three subunits (28Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (87) Google Scholar). The active sites of bGD, yCD, eCDA, and bCDA share virtually identical interaction networks between the attacking water molecule, the zinc ion, the three protein ligands, the base glutamate, and the common moiety of the pyrimidine ring (Fig. 4B). The absolute conservation of a proline residue prior to the conserved cysteine in the CDA superfamily (Pro82 in bGD) may be due to the fixation in orientation of the backbone O (Glu81 O in bGD) for interaction with both the amino group and the leaving ammonium molecule (27Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. Biochemistry. 1997; 36: 4768-4774Crossref PubMed Scopus (82) Google Scholar). Thus, the presence of the conserved signatures HXE (or CXE) and PCXXC in the CDA superfamily indicates a similar zinc-assisted deamination mechanism.Therefore, on the basis of the structural studies on bGD, yCD, and CDAs (2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 27Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. Biochemistry. 1997; 36: 4768-4774Crossref PubMed Scopus (82) Google Scholar), a deamination mechanism for bGD is proposed as outlined in Scheme 1. The nucleobase guanine binds to the active site, and its direct contacts with the C-terminal tail induces the tail to cover the active site entrance, sequestering the reaction from solvent. Glu55 serves as a proton shuttle, abstracting a proton from the zinc-activated water to form the attacking hydroxide ion on the one hand and, on the other hand, protonating the N1 of guanine to form the tetrahedral intermediate. Subsequently, Glu55 also assists the proton transfer from OH2 to NH22 to facilitate the cleavage of the carbon-nitrogen bond. The newly formed xanthine may move toward the zinc ion for ligation, and this would weaken its interaction with the C-terminal tail, allowing its release from the active site.Scheme 1The proposed catalytic mechanism for bGD.View Large Image Figure ViewerDownload (PPT)Structural Divergence in the CDA Superfamily—One of the major variations is the state of protein oligomerization. The dimeric bGD cannot be superimposed on the dimeric yCD and tetrameric bCDA because of the different relative orientations between the subunits. However, all of these deaminases utilize the helical layer(s) and the C-terminal tail for oligomerization. Only one helical layer contributes to the interface in CDAs and yCD, whereas both helical layers are used in bGD because of the domain swapping.A second variation is the C-terminal segment beyond strand β4, which is hypervariable and contributes to substrate recognition through a switching of the active site entrance and/or direct interaction. To date, all of the available structures are in a closed conformation. In bGD, yCD, and bCDA, the C-terminal tail forms a “flap” across the entrance that regulates both substrate binding and product release (2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (87) Google Scholar) (Fig. 3C). However, the active site entrance switch in eCDA is more complicated, perhaps acting through three loops from the adjacent monomer, and remains unclear (1Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (336) Google Scholar). The C-terminal tails in both yCD and bGD contain conserved substrate recognition residues such as Asp155 in yCD and Tyr156 in bGD. These residues seal the active site entrance upon substrate binding and thus limit the size of the substrate binding pocket for the nucleobases. On the other hand, the highly flexible C-terminal tail of bCDA would seem to enlarge the active site cavity to accommodate the larger nucleoside substrate (2Ko T.P. Lin J.J. Hu C.Y. Hsu Y.H. Wang A.H.J. Liaw S.H. J. Biol. Chem. 2003; 278: 19111-19117Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (87) Google Scholar). Neither cytidine nor guanine