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• W1966489764 abstract "Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-Å resolution displayed a tetrameric ring-like structure with an extensive interface of ∼2400 Å2/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure. Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-Å resolution displayed a tetrameric ring-like structure with an extensive interface of ∼2400 Å2/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure. Flavin coenzymes are ubiquitous in all organisms because of their involvements in central metabolic pathways. Plants and many microorganisms obtain the precursor riboflavin by biosynthesis, whereas animals depend on nutritional sources. Numerous pathogenic microorganisms are unable to take up flavins from the environment and hence are absolutely dependent on their endogenous production. Therefore, the enzymes involved in riboflavin biosynthesis have the potential to become attractive candidates for the design of new defenses against antibiotic-resistant pathogens. During riboflavin biosynthesis (1Bacher A. Eberhardt S. Fischer M. Kis K. Richter G. Annu. Rev. Nutr. 2000; 20: 153-167Crossref PubMed Scopus (220) Google Scholar), GTP cyclohydrolase II first catalyzes the hydrolytic C-8 release of GTP to yield formate and pyrophosphate as side products. The product, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (compound 1) is converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate (compound 4) by deamination of the pyrimidine ring and NAD(P)H-dependent reduction of the ribose (Fig. 1). The deamination and reduction steps have been shown to proceed in the opposite order in yeast and Escherichia coli (2Oltmanns O. Bacher A. J. Bacteriol. 1972; 110: 818-822Crossref PubMed Google Scholar, 3Burrows R.B. Brown G.M. J. Bacteriol. 1978; 136: 657-667Crossref PubMed Google Scholar). Most eubacteria contain a bifunctional protein; for instance Bacillus subtilis RibG (BsRibG) 3The abbreviations used are: BsRibG, B. subtilis RibG; BsGD, B. subtilis guanine deaminase; CDA, cytidine deaminase; yCD, yeast cytosine deaminase; T4dCMPD, T4 bacteriophage dCMP deaminase; TAD, tRNA-specific adenosine deaminase; AaTADA, A. aeolicus tRNA-specific adenosine deaminase; DHFR, dihydrofolate reductase; TmDHFR, T. maritima dihydrofolate reductase; AID, activation-induced deaminase; APOBEC, apolipoprotein B mRNA-editing catalytic subunit; r.m.s.d., root-mean-square deviation; AICAR, aminoimidazole-4-carboxamide ribonucleotide.3The abbreviations used are: BsRibG, B. subtilis RibG; BsGD, B. subtilis guanine deaminase; CDA, cytidine deaminase; yCD, yeast cytosine deaminase; T4dCMPD, T4 bacteriophage dCMP deaminase; TAD, tRNA-specific adenosine deaminase; AaTADA, A. aeolicus tRNA-specific adenosine deaminase; DHFR, dihydrofolate reductase; TmDHFR, T. maritima dihydrofolate reductase; AID, activation-induced deaminase; APOBEC, apolipoprotein B mRNA-editing catalytic subunit; r.m.s.d., root-mean-square deviation; AICAR, aminoimidazole-4-carboxamide ribonucleotide. is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain) (4Richter G. Fischer M. Krieger C. Eberhardt S. Lüttgen H. Gerstenschläger I. Bacher A. J. Bacteriol. 1997; 179: 2022-2028Crossref PubMed Google Scholar). In contrast, in fungi, plants and most archaea, these two enzymes are separate (5Baur A. Schaaff-Gerstenschlager I. Boles E. Miosga T. Rose M. Zimmermann F.K. Yeast. 1993; 9: 289-293Crossref PubMed Scopus (14) Google Scholar, 6Fisher M. Romisch W. Saller S. Illarionov B. Richter G. Rohdich F. Eisenreich W. Bacher A. J. Biol. Chem. 2004; 279: 36299-36308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7Graupner M. Xu H. White R.H. J. Bacteriol. 2002; 184: 1952-1957Crossref PubMed Scopus (37) Google Scholar). In yeast, Rib7 is the corresponding reductase, whereas the C-terminal domain of Rib2 is responsible for deamination, in addition to N-terminal pseudouridine synthase activity. The D domain of bacterial RibG was expected to belong to the cytidine deaminase (CDA) superfamily because of the two conserved signatures, H(C)XE and PCX8-9C. The CDA superfamily consists of the mononucleotide deaminases involved in nucleotide metabolism, and the RNA/DNA-editing deaminases involved in gene diversity and in antivirus defense (8Keegan L.P. Leroy A. Sproul D. O'Connell M.A. Genome Biol. 2004; 5: 209-218Crossref PubMed Scopus (117) Google Scholar, 9Harris R.S. Liddament M.T. Nat. Rev. Immunol. 2004; 4: 868-877Crossref PubMed Scopus (524) Google Scholar). The RNA/DNA-editing deaminases include A- to -I tRNA-specific adenosine deaminases (TADs), A- to -I adenosine deaminases acting on RNA, and C- to -U cytidine deaminases acting on RNA/DNA. These deaminases catalyze the hydrolytic deamination of cytosine, guanine, and adenine moieties and several of their therapeutically useful analogues. The available member structures reveal a virtually identical zinc-assisted deamination mechanism with the consensus histidine and cysteines acting as the zinc ligands, whereas the glutamate serves as a proton shuttle (10Ko 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 (99) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 13Almog R. Maley F. Maley G.F. Maccoll R. Van Roey P. Biochemistry. 2004; 43: 13715-13723Crossref PubMed Scopus (23) Google Scholar, 14Kuratani M. Ishii R. Bessho Y. Fukunaga R. Sengoku T. Shirouzu M. Sekine S. Yokoyama S. J. Biol. Chem. 2005; 280: 16002-16008Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, the question of how nature has evolved the CDA fold into these various deaminases requires additional study. Furthermore, the structural fold of the R domain was predicted by 3D-PSSM (15Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1120) Google Scholar) to be similar to dihydrofolate reductase (DHFR). DHFR catalyzes the NADPH-utilizing reduction of dihydrofolate to tetrahydrofolate. Many DHFR inhibitors, such as methotrexate, pyrimethamine, and trimethoprim, have long been used clinically in the treatment of cancer, rheumatoid arthritis, malaria, and bacterial and fungal infection (16Kompis I.M. Islam K Then R.L. Chem. Rev. 2005; 105: 593-620Crossref PubMed Scopus (199) Google Scholar). Therefore, the R domain may become an important target for new drug design. To gain structural insights into the inhibitor design, substrate specificity, and evolution, we have solved the BsRibG structure at 2.41-Å resolution. Protein Preparation—The His-tagged BsRibG was expressed using the pQE30 vector (Qiagen) in E. coli BL21 pLysS. The recombinant protein contains 12 additional vector residues (MRGSH6GS) at the N terminus. One-liter Luria broth (LB) cultures were grown at 37 °C to an A600 of 0.8 and induced with the addition of 1 mm isopropyl β-d-thiogalactopyranoside. Cells were grown for another 5 h at 37°C before harvest. Cell pellets were resuspended in 40 ml of cold buffer A (20 mm Tris-HCl, 200 mm NaCl, pH 7.5) and lysed by French press. After removal of cellular debris by centrifugation at 39,000 × g at 4 °C for 30 min, the supernatant was applied to a 5-ml nickel-nitrilotriacetic acid column (Ni-NTA, Qiagen). The resin was washed with 40 mm imidazole in buffer A, and the protein was eluted with buffer A containing 500 mm imidazole. The protein fractions were collected, dialyzed against buffer B (20 mm Tris-HCl, pH 8.5), and loaded onto a Sepharose™ Q column (Amersham Biosciences). After washing with 230 mm NaCl in buffer B, the BsRibG was eluted with 260 mm NaCl and dialyzed against 20 mm HEPES (pH 7.5) and 5 mm dithiothreitol. Protein Characterization—The enzyme activity assay was carried out as described previously (4Richter G. Fischer M. Krieger C. Eberhardt S. Lüttgen H. Gerstenschläger I. Bacher A. J. Bacteriol. 1997; 179: 2022-2028Crossref PubMed Google Scholar). The substrate was prepared by GTP hydrolysis using the recombinant E. coli GTP cyclohydrolase II. The molecular mass in solution was estimated by a Beckman-Coulter XL-A analytical Ultracentrifuge with an An60Ti rotor. Sedimentation velocity was performed at 20 °C and 40,000 rpm with standard double sector centerpieces. The UV absorption of the cells was scanned every 5 min for 2 h, and the data were analyzed using the SedFit program (17Schuck P. Perugini M.A. Gonzales N.R. Howlett G.J. Schubert D. Biophys. J. 2002; 82: 1096-1111Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Protein Crystallization—The initial crystallization screening was performed with screening kits using the hanging-drop vapor diffusion method at 22 °C. The hanging drops were mixtures of 2 μl of reservoir solution and 2 μl of protein solution. Protein crystals could be obtained under several reservoir solutions, and the best crystals were grown in 26.6% polyethylene glycol 400, 190 mm MgCl2, 5% glycerol, and 95 mm HEPES (pH 7.5) with a protein solution of 20-25 mg/ml. Crystals appeared and reached their final dimensions in 1 week at 15 °C. The NADPH derivative was prepared by soaking crystals for 4 days in reservoir solution containing 10 mm NADPH. X-ray diffraction data were collected and processed at beamlines BL12B2 at SPring-8 (Harima, Japan) and NW12 at the Photon Factory (Tsukuba, Japan). The crystals belong to the P212121 space group with 1 tetramer/asymmetric unit. Structure Determination—The BsRibG structure was determined using single-wavelength anomalous dispersion (SAD) of the endogenous zinc ion. Four Zn2+ positions were identified and refined with SOLVE (18Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar). The initial phase was improved by direct-method phasing refinement using OASIS (19Wang J.W. Chen J.R. Gu Y.X. Zheng C.D. Jiang F. Fan H.F. Terwilliger T.C. Hao Q. Acta Crystallogr. Sect. D. 2004; 60: 1244-1253Crossref PubMed Scopus (32) Google Scholar) before density modification. The initial electron density map showed an interpretable density of ∼70% of the tetramer. About 60% of the initial tetrameric model was automatically built using RESOLVE (18Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (433) Google Scholar). Because the structural fold was predicted to be similar to yeast cytosine deaminase (yCD) and Thermotoga maritima DHFR (TmDHFR) (10Ko 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 (99) Google Scholar, 20Dams T. Auerbach G. Bader G. Jacob U. Ploom T. Huber R. Jaenicke R. J. Mol. Biol. 2000; 297: 659-672Crossref PubMed Scopus (90) Google Scholar), both structures were used to assist in the manual building of the atomic model using TURBO-FRODO (21Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory 81. Silicon Graphics Corp., Mountain View, CA1991Google Scholar), and refinement was carried out against the native data to 2.41-Å resolution using crystallography NMR software (22Brünger 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. 1998; 54: 905-921Crossref PubMed Scopus (16963) Google Scholar). The statistics for data collection and refinement are summarized in Table 1. About 88% of the residues are in the most favored regions of the Ramachandran plot, with the remaining in the additional allowed regions.TABLE 1Statistics for data collection and structural refinementData setNativeZinc-SADNADPHData collectionWavelength (Å)1.00001.28231.0000Unit cell (Å)87.3, 108.4, 186.987.3, 108.5, 187.086.1, 108.0, 186.8Resolution range (Å)50-2.41 (2.50-2.41)50-2.48 (2.57-2.48)50-2.97 (3.08-2.97)Total observations673,346 (48,412)738,284 (55,670)313,672 (22,528)Unique reflections67,740 (6,370)63,545 (5,986)35,885 (3,520)Completeness (%)97.6 (93.2)99.2 (94.9)98.2 (98.1)I/σ <I>17.7 (3.9)22.9 (5.5)18.0 (4.0)Rmerge(%)5.6 (34.9)5.4 (33.5)6.8 (29.4)RefinementResolution range (Å)50-2.41 (2.5-2.41)50-2.97 (3.08-2.97)Reflections (F > 0 σF)67,740 (5,683)35,885 (3,319)Rcryst (%) for 90% data22.6 (28.5)22.4 (32.4)Rfree (%) for 10% data27.7 (31.8)27.0 (37.5)r.m.s. deviationsBond lengths (Å)0.0090.007Bond angles (°)1.441.31Average B-factors (Å2)10,970 protein atoms47.010,970 protein atoms73.04 zinc ions42.24 zinc ions77.0293 water molecules51.8235 water molecules52.648 NADPH atoms84.5 Open table in a new tab Sequence Alignment—To identify the unique signatures for each family member for gene annotation, sequence similarity searches were conducted by PSI-BLAST (23Schäffer 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 (1118) Google Scholar), and multiple sequence alignment of the homologous sequences was performed by ClustalW (24Chenna 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 (4050) Google Scholar). Because of a conservative hydrophobic core and the consensus HXE and PCX2-9C, a structural-based sequence alignment between the CDA members was feasible and was carried out by manual editing according to the available structures. The conserved residues in each family member were mapped onto the known structures to reveal their potential involvement in structural integrity or the enzyme catalysis. Figs. 3, 4, A and B, and 6A were generated by MolScript (25Esnouf R.M. Acta Crystallogr. Sect. D. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar) and Raster3D (26Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3875) Google Scholar), Fig. 5A by BobScript (25Esnouf R.M. Acta Crystallogr. Sect. D. 1999; 55: 938-940Crossref PubMed Scopus (850) Google Scholar), and Fig. 5B by LigPlot (27Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4349) Google Scholar).FIGURE 4Subunit interfaces. Stereo views of the D interface (A) and the R interface (B). Two distinct subunit interfaces are formed by the D and R domains with total buried areas of ∼1300 and ∼3500 Å2, respectively. The D interface is made up mainly of the β2 strand and helices αA and αB, whereas the R interface is made up of the two large loops, LβA-αB and LβF-βG, and the C-terminal tail.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6The NADPH-binding site in the R domain. A, the 2Fo-Fc electron density map for NADPH contoured at 1.5 σ level and is shown in cyan. B, schematic diagram of BsRibG interactions with the NADPH cofactor. Hydrogen bonds are presented as dashed lines; the interatomic distances are given in angstroms. “Radiating” spheres indicate hydrophobic contacts between the cofactor and the surrounding residues.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Structural conservation and divergence of the CDA superfamily. A, stereo view of structural superposition of BsRibG (red), yCD (blue) (10Ko 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 (99) Google Scholar), T4dCMPD (green) (13Almog R. Maley F. Maley G.F. Maccoll R. Van Roey P. Biochemistry. 2004; 43: 13715-13723Crossref PubMed Scopus (23) Google Scholar), and AaTADA (yellow) (14Kuratani M. Ishii R. Bessho Y. Fukunaga R. Sengoku T. Shirouzu M. Sekine S. Yokoyama S. J. Biol. Chem. 2005; 280: 16002-16008Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The zinc ion is displayed as a sphere (magenta) with the yCD inhibitor 3,4-dihydrouracil (DHU)(cyan) shown as ball- and -stick representations. These deaminases share the conserved β-sheet and helices αA-αC and even a part of the αE helix. T4dCMPD contains a ∼60-residue insertion, which folds into two helices (αB′ and αB″) and flexibleloops. The C-terminal tail of yCD folds backward to limit the pocket size, whereas those of the remaining members swing away to enlarge the active-site cavity. B, superposition of the active sites of BsRibG (magenta), yCD (cyan), and T4dCMPD (green). The residue numbering is labeled in the same color for each protein. The deaminases display highly conserved interaction networks surrounding the target amino group of the nucleobase ring. In contrast, each member contains its own unique substrate recognition residues. C, multiple sequence alignment of some CDA members. The Arabidopsis thaliana deaminase (At363) (6Fisher M. Romisch W. Saller S. Illarionov B. Richter G. Rohdich F. Eisenreich W. Bacher A. J. Biol. Chem. 2004; 279: 36299-36308Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) involved in riboflavin biosynthesis is also included. The GenBank™ accession codes are listed in the right column. Secondary structure elements for BsRibG are labeled (s.s). The number of residues in gaps is indicated in parentheses, and the protein length is in brackets. The superfamily signatures, H(C)XE and PCX2-9C, are shaded in cyan, whereas the residues for the conserved hydrophobic core are in yellow. In addition, the substrate-binding residues in the known complex structures are shaded in red, and those that are predicted are in blue. The unique signatures for each member are highlighted in italics, and the residues involved in loss-of-function point mutants of AID are shaded in magenta.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overall Structure—Analytical ultracentrifugation experiments clearly demonstrated that BsRibG exists as a tetramer in solution as well as in crystal form, where the enzyme forms a tetrameric ring-like structure (Figs. 2 and 3a). The current tetrameric model contains residues 1-359 for each subunit. Each monomer is composed of two separate functional domains (Fig. 3b). The N-terminal D domain (residues 1-143) consists of a central five-stranded β-sheet (β1-β5) with β1 running antiparallel to the others. The β-sheet is sandwiched by two helices (αA and αE) on one side and by three helices (αB, αC, and αD) on the other side. The C-terminal helix αF extends away from the D domain and connects to the R domain. The R domain (residues 146-359) is composed of a large nine-stranded β-sheet (βA-βH and βD′) with the C-terminal strand βH running antiparallel to the others. The β-sheet is flanked by five α-helices (αB, αC, αD′, αE, and αF). The secondary structure elements of the R domain are numbered as for DHFRs (28Schnell J.R. Dyson H.J. Wright P.E. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 119-140Crossref PubMed Scopus (400) Google Scholar). The four subunits in the crystal asymmetric unit did not show significant differences between each individual domain except for several loops (root-mean-square deviations (r.m.s.d.) of 0.38-0.46 and 0.59-0.73 Å for the backbones of residues 2-139 and residues 146-359, respectively). However, the relative orientations between the D and R domains are slightly different, resulting in a weak noncrystallographic symmetry. Molecules A and B interact with each other through their D domains with a buried surface area of ∼650 Å2 per D domain (the D interface) (Fig. 4A), whereas molecule A makes extensive contacts with molecule C through their respective R domains with a buried area of ∼1750 Å2 per R domain (the R interface) (Fig. 4B). There are no contacts between molecules A and D in the tetramer. The D interface is made up mainly of the N-terminal two helices αA and αB, the β2 strand, and the connected loops (residues 4-19 and 35-61). There are 14 direct hydrogen bonds between the protein atoms across the interface and hydrophobic patches formed by Leu8, Leu12, Ile36, Met39, Leu43, and Met57. The anti-parallel disposition of the two pseudodyad-related αA helices also contributes to the interface by dipole-dipole interactions. Interestingly, the side chains of His56 from the two D domains stack very well, with a distance of 3.3-3.4 Å between the aromatic rings. The R interface mainly is made up of: the two large loops, LβA-αB (residues 156-169) and LβF-αG (residues 314-338); and the C-terminal residues 339-358. There are 18 direct hydrogen bonds between the protein atoms across the interface, and extensive hydrophobic patches are by Try169 formed, Pro314, Lys315, Leu316, Ile317, Leu325, Phe331, Met334, Val337, Leu339, Leu340, Phe342, Ile345, Ile352, and Leu354. Structural Conservation in the CDA Superfamily—As expected, the D domain displays high structural homology to the available structures of the CDA members including yCD, B. subtilis guanine deaminse (BsGD), CDAs, T4 bacteriophage dCMP deaminse (T4dCMPD), and Aquifex aeolicus TADA (AaTADA), and subdomains 2 and 4 of the chicken AICAR transformylase domain (10Ko 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 (99) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar, 13Almog R. Maley F. Maley G.F. Maccoll R. Van Roey P. Biochemistry. 2004; 43: 13715-13723Crossref PubMed Scopus (23) Google Scholar, 14Kuratani M. Ishii R. Bessho Y. Fukunaga R. Sengoku T. Shirouzu M. Sekine S. Yokoyama S. J. Biol. Chem. 2005; 280: 16002-16008Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29Wolan D.W. Greasly S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar). The TADAs in prokaryotes and the TAD2/TAD3 heterodimers in eukaryotes are responsible for the I34 alteration at the wobble position of the tRNA anticodon, whereas TAD1 creates the unique 1-methyl-I37 in eukaryotic tRNAAla (8Keegan L.P. Leroy A. Sproul D. O'Connell M.A. Genome Biol. 2004; 5: 209-218Crossref PubMed Scopus (117) Google Scholar). Detailed structural comparisons reveal a common three-layer α/β/α structure, in which five β-strands (β1-β5) and three helices (αA-αC) correspond closely, whereas the remainder varies across the different deaminases (Fig. 5A). The main chain atoms of the 65-70 structurally equivalent residues are overlaid with an r.m.s.d. of 0.75 to 1.35 Å and with 8-24% sequence identity. These structural elements in the CDA fold form a conservative hydrophobic core, which is also preserved in the AICAR transformylase domain, implying that the hydrophobic core has been highly conserved throughout evolution. The active site of the D domain contains one tightly bound endogenous zinc ion, for which the anomalous data provided sufficient phase information for structure determination. The zinc ion is tetrahedrally coordinated by His49 Nϵ1 (2.0 Å), Cys74 Sγ (2.4 Å), Cys83 Sγ (2.3 Å), and a water molecule (2.0 Å).The zinc-bound water molecule interacts with Glu51 Oϵ2 (2.5 Å). The active-site architecture resembles those of the CDA members, which share a similar zinc-assisted deamination mechanism with a virtually identical interaction network between the common moiety of the pyrimidine ring of the substrate, the zinc ion, the zinc-bound water molecule, the zinc ligands, and the base glutamate (Fig. 5B). In addition, the active-site cavities of these deaminases are mainly made up of the C-terminal tail and the loops connecting the αA-β1, β2-αB, β3-αC, and β4-αD (Fig. 5C). Based on the structural comparison of the active-site cavities, the substrate of the D domain was modeled into the active site through superposition of the nucleobase rings because of the highly conserved interaction networks surrounding the target amino group (Fig. 5B). The model was then subjected to energy minimization with crystallography NMR software. Simulation of the complex structure suggested that the nucleophilic OH-2 group of the pyrimidine ring coordinates to the catalytic zinc ion and interacts with Glu51Oϵ2 and Cys74 N. The NH-3 group hydrogen bonds with Glu51 Oϵ1, the O-4 atom with Ala50 N and His42 Nδ, and the NH2-5 with Asn23 Oδ1 and His42 Nδ1. The two hydroxyl groups of the ribose have close contacts with the side chains of Asp101 and Asn103. The phosphate moiety forms salt bridges with His76 and Lys79, located in the unique insertion between the two zinc ligand cysteines, and these interactions are essential for the deamination activity because the enzyme cannot utilize the dephosphorylated form as substrate (3Burrows R.B. Brown G.M. J. Bacteriol. 1978; 136: 657-667Crossref PubMed Google Scholar). These predicted substrate-binding residues are all highly conserved in the eubacterial RibGs. However, the fungal deaminases such as yeast Rib2 (yRib2) apparently contain different substrate-interacting residues because of their distinct substrate, which has an open ribityl group instead of a cyclic ribose (Figs. 1 and 5C). Structural Divergence in the CDA Superfamily—The CDA members exist as an oligomer. All of the available member structures except for BsRibG utilize helices αB-αD and surrounding loops for oligomerization. BsGD, yCD, and AaTADA display similar dimeric structures (10Ko 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 (99) Google Scholar, 11Liaw S.H. Chang Y.J. Lai C.T. Chang H.C. Chang G.G. J. Biol. Chem. 2004; 279: 35479-35485Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Kuratani M. Ishii R. Bessho Y. Fukunaga R. Sengoku T. Shirouzu M. Sekine S. Yokoyama S. J. Biol. Chem. 2005; 280: 16002-16008Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The swapping of the C-terminal segment in BsGD causes additional dimeric contacts including the β5 strand and helices αA and αE. CDAs form tetramers in which one subunit interacts with the other three subunits (12Johansson E. Mejlhede N. Neuhard J. Larsen S. Biochemistry. 2002; 41: 2563-2570Crossref PubMed Scopus (90) Google Scholar). The hexameric T4dCMPD contains two types of intersubunit interfaces (13Almog R. Maley F. Maley G.F. Maccoll R. Van Roey P. Biochemistry. 2004; 43: 13715-13723Crossref PubMed Scopus (23) Google Scholar). In contrast, the D interface of BsRibG is very distinct from the others and possesses the fewest contacts, with a total buried area of ∼1300 Å2 (Fig. 4A). In addition to the loops and helices αA and αB, RibG includes the β2 strand in the D interface. The distinct intersubunit orientation in the D interface also separates the active sites away from each other with an inter-zinc distance of 30 Å. Notably, the shortest inter-zinc distance in the other five deaminases is about 14-15 Å. The C-t" @default.
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• W1966489764 title "Crystal Structure of a Bifunctional Deaminase and Reductase from Bacillus subtilis Involved in Riboflavin Biosynthesis" @default.
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