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• W2032393676 abstract "S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs. S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs. S-Adenosylmethionine decarboxylase (AdoMetDC) 1The abbreviations used are: AdoMetDC, S-adenosylmethionine decarboxylase; AdoMet, S-adenosylmethionine; dcAdoMet, decarboxylated S-adenosylmethionine; HisDC, histidine decarboxylase; AspDC, asparate-α-decarboxylase; ArgDC, arginine decarboxylase; PLP, pyridoxal-5′-phosphate; MAD, multiple wavelength anomalous dispersion; IPTG, isopropyl β-d-thiogalactoside; NCS, noncrystal-lographic symmetry; MeAdoMet, S-adenosylmethionine methyl ester; r.m.s.d., root mean square deviation; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: AdoMetDC, S-adenosylmethionine decarboxylase; AdoMet, S-adenosylmethionine; dcAdoMet, decarboxylated S-adenosylmethionine; HisDC, histidine decarboxylase; AspDC, asparate-α-decarboxylase; ArgDC, arginine decarboxylase; PLP, pyridoxal-5′-phosphate; MAD, multiple wavelength anomalous dispersion; IPTG, isopropyl β-d-thiogalactoside; NCS, noncrystal-lographic symmetry; MeAdoMet, S-adenosylmethionine methyl ester; r.m.s.d., root mean square deviation; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is a critical regulatory enzyme in the polyamine biosynthetic pathway in all species and has been the subject of biochemical studies spanning several decades using microorganisms, plants, and mammalian systems (reviewed in Refs. 1Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3214) Google Scholar, 2Pegg A.E. Xiong H. Feith D. Shantz L.M. Biochem. Soc. Trans. 1998; 26 (26): 580-586Crossref PubMed Scopus (97) Google Scholar, 3Franceschetti M. Hanfrey C. Scaramagli S. Torrigiani P. Bagni N. Burtin D. Michael A.J. Biochem. J. 2001; 353: 403-409Crossref PubMed Scopus (85) Google Scholar). AdoMetDC catalyzes the removal of the carboxylate group from S-adenosylmethionine (AdoMet) to form S-adenosyl-5′-(3-methylthiopropylamine) (dcAdoMet), which is committed to act as the n-propylamine group donor for the synthesis of the polyamines spermine and spermidine from the diamine putrescine. AdoMetDC is a crucial control point within the polyamine pathway and its activity is highly regulated during the cell cycle. These polyamines have been shown to be involved in the initiation and maintenance of proliferative states and are essential for cell growth and differentiation (4Pegg A.E. Biochem. J. 1986; 234: 249-262Crossref PubMed Scopus (1424) Google Scholar, 5Wallace H.M. Fraser A.V. Hughes A. Biochem. J. 2003; 376: 1-14Crossref PubMed Scopus (758) Google Scholar). AdoMetDC belongs to a small class of decarboxylating enzymes that use a covalently bound pyruvate as a prosthetic group rather than the cofactor pyridoxal 5′-phosphate (PLP) typically employed in amino acid decarboxylation reactions (6van Poelje P.D. Snell E.E. Annu. Rev. Biochem. 1990; 59: 29-59Crossref PubMed Scopus (199) Google Scholar, 7Hackert M.L. Pegg A.E. Sinnott M.L. Comprehensive Biological Catalysis. Academic Press, London1997: 201-216Google Scholar). All AdoMetDCs currently characterized are pyruvoyl enzymes but they can be divided into two classes. Class 1 enzymes are present in bacteria and Archaea, and class 2 enzymes are present in Eukarya (see Table I for representative members of each class). Formation of the active enzyme in both cases involves a self-maturation process in which the active site pyruvoyl group is generated from an internal serine residue via an autocatalytic post-translational modification. Two non-identical subunits are generated from the proenzyme in this reaction, and the pyruvate is formed at the N terminus of the α-subunit, which is derived from the carboxyl end of the proenzyme. The post-translation cleavage follows an unusual pathway, termed nonhydrolytic serinolysis, in which the side chain hydroxyl group of the serine supplies its oxygen atom to form the C terminus of the β-chain, while the remainder of the serine residue is converted to ammonia and the pyruvoyl group blocking the N terminus of the α-chain. Although all AdoMet-DCs undergo the same self-maturation process, the class 1 and class 2 enzymes have almost no detectable sequence homology, and they do not share similarity to any of the other known pyruvoyl-dependent amino acid decarboxylases.Table IRepresentative AdoMetDCsSourceProenzymeResidues in α-subunitaNot including the pyruvate at its N terminusResidues in β-subunitOligomer structureSequence at cleavage sitebThe serine that forms the pyruvate is shown in bold textkDaClass IAE. coli30152111(αβ)4VVAHLDKS112HICVHC. acetobutylicum32155118Not knownVLAHLDKS119HITVHClass IBT. maritima156762(αβ)2GVVVISES63HLTIHM. jannaschii146063(αβ)2GVAVLAES64HIAIHB. subtilis146264Not knownGVVIISES65HLTIHClass IIHuman3826667(αβ)2EAYVLSES68SMFVSPotato4028772αβDSYVLSES73SLFVYT. cruzi4228485Not knownRSYVLTES86SLFVMYeast4630887Not knownDAFLLSES88SLFVFa Not including the pyruvate at its N terminusb The serine that forms the pyruvate is shown in bold text Open table in a new tab The class 1 AdoMetDCs can be further divided into two groups. Class 1A AdoMetDCs are found primarily in Gram-negative bacteria as the speD gene product. For example, the Escherichia coli enzyme cleaves to give an 18-kDa α-chain and a 12-kDa β-chain (8Markham G.D. Tabor C.W. Tabor H. J. Biol. Chem. 1982; 257: 12063-12068Abstract Full Text PDF PubMed Google Scholar, 9Diaz E. Anton D.L. Biochemistry. 1991; 30: 4078-4081Crossref PubMed Scopus (20) Google Scholar). The active form of the enzyme is an (αβ)4 tetramer and requires a divalent metal ion, such as Mg2+, for catalytic activity. Class 1B AdoMetDCs have been identified in some Gram-positive bacteria (10Sekowska A. Coppée J.-Y. Le Caer J.-P. Martin-Verstraete I. Danchin A. Mol. Microbiol. 2000; 36: 1135-1147Crossref PubMed Scopus (34) Google Scholar) and Archeabacteria (11Kim A.D. Graham D.E. Seeholzer S.H. Markham G.D. J. Bacteriol. 2000; 182: 6667-6672Crossref PubMed Scopus (22) Google Scholar). Based on the studies with the proenzymes from Bacillus subtilis (10Sekowska A. Coppée J.-Y. Le Caer J.-P. Martin-Verstraete I. Danchin A. Mol. Microbiol. 2000; 36: 1135-1147Crossref PubMed Scopus (34) Google Scholar) and Methanococcus jannaschii (11Kim A.D. Graham D.E. Seeholzer S.H. Markham G.D. J. Bacteriol. 2000; 182: 6667-6672Crossref PubMed Scopus (22) Google Scholar), the class 1B AdoMetDCs cleave to form α- and β-chains, each with a molecular mass of about 7 kDa. This class forms an (αβ)2 dimer and does not require Mg2+ or other activators. Class 1A and 1B enzymes show low levels of sequence similarity. These similarities are evident in the residues surrounding the probable cleavage site and those surrounding a cysteine residue, which was identified as part of the active site from modifications that occur during substrate-mediated inactivation of the E. coli and Salmonella typhimurium enzymes (9Diaz E. Anton D.L. Biochemistry. 1991; 30: 4078-4081Crossref PubMed Scopus (20) Google Scholar, 12Li Y.F. Hess S. Pannell L.K. Tabor C.W. Tabor H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10578-10583Crossref PubMed Scopus (25) Google Scholar). Crystal structures are now available for several pyruvoyl enzymes including histidine decarboxylase (HisDC) from Lactobacillus 30a (13Gallagher T. Snell E.E. Hackert M.L. J. Biol. Chem. 1989; 264: 12737-12743Abstract Full Text PDF PubMed Google Scholar, 14Gallagher T. Rozwarski D.A. Ernst S.R. Hackert M.L. J. Mol. Biol. 1993; 230: 516-528Crossref PubMed Scopus (50) Google Scholar), aspartate decarboxylase (AspDC) from E. coli (15Albert A. Dhanaraj V. Genschel U. Khan G. Ramjee M.K. Pulido R. Sibanda B.L. von Delft F. Witty M. Blundell T.L. Smith A.G. Abell C. Nat. Struct. Biol. 1998; 5: 289-293Crossref PubMed Scopus (88) Google Scholar, 16Schmitzberger F. Kilkenny M.L. Lobley C.M. Webb M.E. Vinkovic M. Matak-Vinkovic D. Witty M. Chirgadze D.Y. Smith A.G. Abell C. Blundell T.L. EMBO J. 2003; 22: 6193-6204Crossref PubMed Scopus (47) Google Scholar), arginine decarboxylase (ArgDC) from M. jannaschii (17Tolbert W.D. Graham D.E. White R.H. Ealick S.E. Structure. 2003; 11: 285-294Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and class 2 AdoMetDCs from human (18Ekstrom J.E. Matthews I.I. Stanley B.A. Pegg A.E. Ealick S.E. Structure. 1999; 7: 583-595Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Ekstrom J.L. Tolbert W.D. Xiong H. Pegg A.E. Ealick S.E. Biochemistry. 2001; 40: 9495-9504Crossref PubMed Scopus (49) Google Scholar, 20Tolbert D.W. Ekstrom J.L. Mathews I.I. Secrist J.A.I. Kapoor P. Pegg A.E. Ealick S.E. Biochemistry. 2001; 40: 9484-9494Crossref PubMed Scopus (54) Google Scholar) and potato (21Bennett E.M. Ekstrom J.L. Pegg A.E. Ealick S.E. Biochemistry. 2002; 41: 14509-14517Crossref PubMed Scopus (29) Google Scholar). Although all of these enzymes generate the pyruvoyl group by internal serinolysis, only ArgDC and HisDC are homologous to each other (17Tolbert W.D. Graham D.E. White R.H. Ealick S.E. Structure. 2003; 11: 285-294Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The three-dimensional structure of the human AdoMetDC demonstrated that its active site is located far from the interface between the two αβ protomers and that it utilizes residues from both the α- and β-chains (18Ekstrom J.E. Matthews I.I. Stanley B.A. Pegg A.E. Ealick S.E. Structure. 1999; 7: 583-595Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 20Tolbert D.W. Ekstrom J.L. Mathews I.I. Secrist J.A.I. Kapoor P. Pegg A.E. Ealick S.E. Biochemistry. 2001; 40: 9484-9494Crossref PubMed Scopus (54) Google Scholar). The topology of each αβ protomer showed an internal structural duplication in which residues 4–164, which contain the pyruvoyl group, and residues 172–329 have similar topologies. The two halves of the human AdoMetDC protomer are similar in size to the class 1 protomer; however, this is the only indication that the two classes of pyruvoyl-dependent AdoMetDC might be structurally homologous. Here we report the structures of the wild-type proenzyme and S63A mutant of a class 1B AdoMetDC from T. maritima determined to 1.55 and 1.7 Å resolution, respectively, using selenomethionine multiwavelength anomalous diffraction (MAD) phasing methods. The class 1B TmAdoMetDC structure provides striking evidence of an ancient gene duplication event resulting in the class 2 AdoMetDC enzymes. TmAdoMetDC is synthesized as a 14.8 kDa proenzyme, which after processing contains two chains, β and α, of molecular mass 7.0 and 7.8 kDa, respectively. The active form of TmAdoMetDC is an (αβ)2 dimer. Each protomer contains one active site that occurs at the dimer interface and also requires residues from the adjacent protomer. Several key active site residues involved in substrate binding, catalysis, or proenzyme processing that were identified in the human and potato class 2 AdoMetDCs are readily recognized in the class 1B TmAdoMetDC despite the overall low primary sequence homology. A sequence alignment of EcAdoMetDC with TmAdoMetDC shows 13% identity. Homology modeling was carried out for the core structure of EcAdoMetDC based on the limited sequence homology and a structural superposition of the T. maritima and human AdoMetDCs. Cloning of T. maritima speD—PCR was performed using T. maritima genomic DNA (purchased from ATCC) as the template with the following primer pair: TmSpeDF: TAG TAG CAT ATG AAG AGT CTG GGA AGG CAC (inserts an NdeI site at the start of the gene) and TmSpeDR: TAG TAG CTC GAG TCA GAC GGC GGC CTT GTG CGG (inserts an XhoI site after the stop codon of the gene). The amplified PCR product was purified (QIAquick PCR purification kit from Qiagen), and ligated into pET-28a. A representative clone was sequenced and named pTmSpeD.28. Mutagenesis of T. maritima speD—Site-directed mutagenesis with the QuikChange kit was performed according to the manufacturer's guidelines. The following complementary primer pair was used: TmS-peDSAF: GGT GGT GAT ATC TGA AGC TCA CCT AAC CAT TCA CAC CTG GCC and TmSpeDSAR: GGC CAG GTG TGA ATG GTT AGG TGA GCT TCA GAT ATC ACC ACC. Clones were screened by restriction digest for the introduction of an AluI site. A representative clone with the correct restriction pattern was sequenced and named pTmSpeD.28 S63A. Protein Expression and Purification—For the production of native S63A protein, the pTmSpeD.28 S63A plasmid was transformed into the BL21Star(DE3)pRare strain of E. coli (Invitrogen). A 5-ml saturated starter culture was used to inoculate 1 liter of LB supplemented with 15 μg/ml chloramphenicol and 30 μg/ml kanamycin. The cells were grown at 37 °C until they reached an OD600 of 0.4, at which point the temperature was shifted to 15 °C. The cells were induced with 1 mm isopropyl β-d-thiogalactoside (IPTG) at an OD600 of ∼0.6. After induction for 16 h the cells were spun down at 5,000 rpm for 10 min and stored at -80 °C. For production of the S63A TmAdoMetDC mutant protein with selenomethionine (SeMet) incorporated, the methionine auxotrophic strain of E. coli, B834(DE3) (Novagen), was transformed with pTm-SpeD.28 S63A. The growth medium contained M9 salts supplemented with all amino acids (40 μg/ml each) except l-methionine, which was replaced with l-SeMet, 0.4% (w/v) glucose, 2 mm MgSO4, 25 μg/ml FeSO4·7H2O,1mm CaCl2,35 μg/ml kanamycin, and a 1% BME vitamin solution (Invitrogen). The cells from the initial 5-ml starter culture were washed with the above medium, and used to start a 1-liter culture. This culture was grown to an OD600 ∼0.6, at which point the temperature was lowered to 25 °C, and the cells were induced with 500 μm IPTG. After induction for 5 h, the cells were spun down and stored at -80 °C. For production of the TmAdoMetDC proenzyme, pTmSpeD.28 was transformed into the B834(DE3) strain of E. coli (Novagen). The cells were grown using LB media supplemented with 35 μg/ml kanomycin. The remainder of the growth protocol was as described for the SeMet S63A protein. All purification steps were carried out at room temperature. The cells were resuspended in 35 ml of binding buffer (50 mm Tris-HCl, pH 8.0, 10 mm imidazole, 500 mm NaCl) and lysed using a French press. The crude extract was centrifuged, and the resulting supernatant was stirred for 1 h with 1.5 ml of Ni-NTA beads (Novagen) equilibrated with the binding buffer. The resin was then loaded into a polypropylene column and washed with binding buffer (100 ml) followed by 50 ml of wash buffer (50 mm Tris HCl, pH 8.0, 35 mm imidazole, 500 mm NaCl). S63A AdoMetDC was eluted from the column with 15 ml of elution buffer (50 mm Tris HCl, pH 8.0, 150 mm imidazole, 500 mm NaCl). After dialysis with 20 mm Tris-HCl, pH 8.0 and 1 mm dithiothreitol, the protein was concentrated to 6 mg/ml, frozen, and stored at -80 °C. The purification protocol for the wild-type TmAdoMetDC was as described for the S63A protein. The wild-type TmAdoMetDC was found to be greater than 95% unprocessed as determined by Coomassie-stained SDS-PAGE (data not shown). Processing of TmAdoMetDC—The proenzyme derived from the Tm-speD gene was synthesized in vitro using the Promega TnT system (catalog L4610). Protein was translated from the pTmSpeD.28 or the same plasmid carrying mutants of TmSpeD (S55A, S63A, H68A, or C83A). The TnT reaction was carried out at 30 °C for 1 h according to manufacturer's instructions with 1 μg of DNA used/50 μl of reaction. Processing was then induced by heating at 65 °C. Aliquots of 5 μl of the TnT reaction product were diluted with 10 μl of 0.075 m sodium phosphate buffer, pH 6.8, and the tubes were transferred to a heat block pre-equilibrated to 65 °C. At the end of the desired processing time, 15 μl of Tricine gel loading buffer (4% SDS, 12% glycerol, 2% β-mercaptoethanol, 50 mm Tris, 0.01% Coomassie Brilliant Blue G) was added to each tube to stop the reaction, and the tube was placed on ice. As soon as the tube had cooled, it was placed at -20 °C. When all samples had been obtained, the samples were thawed, boiled 5 min, microcentrifuged briefly, and loaded onto 12% Tricine gels. Gels were run at 45 mA/gel and stopped when the dye front was just at the bottom of the gel. Gels were then fixed in 10% acetic acid, 30% MeOH in H2O. Fixative was changed twice at 15-min intervals and then gels were left overnight (12–16 h) in fixative on a gently rocking platform. After fixing, the gels were dried on a gel drier for 2–2.5 h at 80 °C and then placed on a phosphorimager screen for visualization. Visualization was carried out on a Molecular Dynamics PhosphorImager 595, and quantitation was done with ImageQuant software. In some experiments, at the end of the 65 °C incubation to induce processing, the tube was cooled and 15 μl of 1 m hydroxylamine hydrochloride was added. The sample was then incubated at 37 °C for the specified time, and the gel-loading buffer was added at the end of the 37 °C incubation. Crystallization—Small initial crystals of the native S63A TmAdoMetDC were obtained using the Crystal Screen™ sparse matrix kit (22Jancarik J. Kim S.H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2076) Google Scholar). The optimized crystallization conditions were found to be 2.0–2.4 m ammonium formate, 100 mm HEPES, pH 8.0, using the hanging-drop method at room temperature. Crystals appeared after 2 weeks and reached their maximum size of 0.15 mm × 0.1 mm × 0.1 mm within 6 weeks. The best SeMet-S63A protein crystals grew under similar conditions with an alkaline shift to pH 8.6 and an increase in precipitant concentration to 3.2 m. Under these conditions S63A TmAdoMetDC crystallizes in the trigonal space group R3 with unit cell dimensions of a = 104.97 Å and c = 69.52 Å. Each asymmetric unit contains a complete dimer with a solvent content of 52%. Crystals of the wild-type TmAdoMetDC proenzyme were obtained under similar conditions to the S63A mutant TmAdoMetDC. The protein concentration was 5 mg/ml, and the optimized well solution contained 3.2 m ammonium formate and 100 mm HEPES, pH 7.8. For cryoprotection, the crystals were transferred to a stabilization solution similar to the mother liquor, but with the ammonium formate concentration increased by 200 mm. The stabilization solution was gradually replaced with a cryoprotectant solution containing increasing concentrations of glycerol (5% steps until the final concentration of 20% was reached). The crystals were frozen by plunging them into liquid nitrogen and stored for later use. Data Collection and Processing—The diffraction experiments were performed at beamline 8-BM at the Advanced Photon Source using a Quantum 315 detector in binned format. A three-wavelength data set was collected to 2.1 Å resolution on the SeMet-S63A TmAdoMetDC. Following calibration of the beam using a Se foil, a fluorescence scan was taken on a SeMet-TmAdoMetDC crystal. For data collection, one wavelength was chosen at the maximum of f ′ (edge), another was chosen at the maximum of f ″ (peak) and a high energy remote wave-length was chosen 200 eV above the edge. Data were collected over 120° using 15 s for each 1° oscillation with a crystal to detector distance of 280 mm. Bijvoet pairs were measured after each 40° wedge using inverse beam geometry. A native S63A data set was collected over a range of 180° using 40 s for each 1° oscillation at a crystal to detector distance of 220 mm. A wild-type proenzyme data set was collected over a range of 120° using a 17 s exposure time for each 0.5° oscillation at a crystal to detector distance of 200 mm. The HKL2000 suite (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar) of programs was used for integration and scaling of all data sets. The data collection statistics are summarized in Table II. Ambiguity in space group assignment between R3 and R32 indicated a potential twinning problem. Cumulative intensity distribution analysis and analysis of second moments indicated twinning for all data sets. The merohedral twin fraction was estimated at 0.25, 0.42, and 0.28, respectively, for the native S63A, SeMet-S63A, and wild-type proenzyme TmAdoMetDC data sets using the twinning server (24Yeates T.O. Methods Enzymol. 1997; 276: 344-358Crossref PubMed Scopus (356) Google Scholar).Table IISummary of data collection and processing statisticsWild typeS63ASeMet S63APeakInflectionRemoteWavelength (Å)0.97860.96410.97920.97930.9641Space groupR3R3R3R3R3a = b = (Å)104.99104.97104.90104.94104.99c = (Å)69.6969.5269.5869.6069.63Resolution (Å)1.551.72.02.12.1Unique reflections4056731186192881930716683RedundancyaValues for the outer resolution shell are given in parentheses3.7 (3.4)5.7 (5.6)7.5 (7.1)7.5 (6.3)7.5 (6.9)% completeaValues for the outer resolution shell are given in parentheses98.2 (98.2)99.8 (100)99.9 (99.8)99.9 (99.9)99.9 (99.9)I/σaValues for the outer resolution shell are given in parentheses29.3 (3.1)30.7 (4.7)29.8 (4.5)28.3 (5.2)29 (5.1)RsymaValues for the outer resolution shell are given in parentheses, bRsym = ΣΣi|Ii - 〈I〉|/Σ〈I〉, where 〈I〉 is the mean intensity of N reflections with intensities Ii and common indices h, k, and l.4.0 (33.9)4.8 (38.5)5.5 (42.0)6.1 (38.9)6.2 (42.5)a Values for the outer resolution shell are given in parenthesesb Rsym = ΣΣi|Ii - 〈I〉|/Σ〈I〉, where 〈I〉 is the mean intensity of N reflections with intensities Ii and common indices h, k, and l. Open table in a new tab MAD Phasing—The initial selenium atom positions of the two non-terminal methionine residues were determined utilizing the iterative Patterson search technique as implemented in SOLVE (25Terwilliger T. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar) with untreated twinned MAD data to 2.7 Å resolution. The initial phases from SOLVE were improved by density modification using RESOLVE (25Terwilliger T. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar) yielding an overall figure of merit of 0.72. Model Building and Structure Refinement—All model building was performed using the computer graphics program O (26Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). The two monomers of the S63A dimer were built by tracing a polyalanine model through clear stretches of backbone density. From the initial model an approximate 2-fold non-crystallographic symmetry (NCS) axis was identified. The map was further improved through NCS averaging and density modification with a protein mask that was created from residues 10–115 and 170–285 of the human AdoMetDC using CNS (27Brünger 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 (16919) Google Scholar). The modified map showed improved connectivity and clear side chain electron density, which allowed the sequence alignment and connectivity to be deciphered. However, several segments of the protein were missing in the experimental electron density; the initial model was about 75% complete. Refinement of the initial model against the 1.7 Å native data set was carried out in CNS using the protocols for twinned data. The refinement procedure involved successive rounds of simulated annealing refinement, temperature factor refinement, and model rebuilding. The S63A structure minus water molecules and three residues before and after the site of pyruvate formation was used as the molecular replacement model for the wild-type proenzyme structure. Molecular replacement was carried out in CNS (27Brünger 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 (16919) Google Scholar) using all data between 3.4 and 10 Å. Model building and refinement were carried out as for the S63A structure. The final refinement statistics are shown in Table III.Table IIIRefinement statistics and model buildingWild typeS63AResolution (Å)1.551.7Total no. of non-hydrogen atoms2,1121,997No. of protein atoms1,9501,884No. of water oxygen atoms162113No. of reflections in refinement39,87327,853No. of reflections in the test set3,8322,954R-factoraR-factor = Σhkl‖Fobs| - k|Fcalc‖/Σhkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively (%)15.014.8RfreebFor Rfree, the sum is extended over a 10% subset of reflections excluded from all stages of refinement (%)18.518.6Refined twin fraction0.280.273R.m.s. deviation from ideal geometryBonds (Å)0.0060.006Angles (°)1.2001.285Ramachandran plotMost favored (%)92.991.9Allowed (%)7.18.1Generously allowed (%)0.00.0Disallowed (%)0.00.0Average B-factors (Å)227.328.6Main chain25.225.3Side chain27.829.2Solvent39.642.9a R-factor = Σhkl‖Fobs| - k|Fcalc‖/Σhkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectivelyb For Rfree, the sum is extended over a 10% subset of reflections excluded from all stages of refinement Open table in a new tab EcAdoMetDC Modeling—Templates for a homology model of the E. coli enzyme were chosen from the known AdoMetDC structures. The S68A human AdoMetDC structure (PDB code 1MSV) was separated into two templates corresponding to the N- and C-terminal domains. The highest sequence similarity with EcAdoMetDC was 19% for the TmAdoMetDC sequence (13% identity). The β-sheet domains of the TmAdoMetDC and the N- and C-terminal halves of the S68A human AdoMetDC were structurally superimposed using LSQ (28Kleywegt G.J. Jones T.A. Macromol. Crystallogr., Pt. B. 1997; 277: 525-545Crossref Scopus (303) Google Scholar), and a ClustalW alignment (29Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar). The sequence alignment of the three templates with EcAdoMetDC were manually adjusted based on the structural superposition. Ten homology models were built from the three template structures using Modeler version 6 (30Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10294) Google Scholar, 31Marti-Renom M.A. Yerkovich B. Sali A. Coligan J.E. Comparative Protein Structure Prediction, Current Protocols in Protein Science. John Wiley & Sons, Inc., New York2002: 2.9.1-2.9.22Google Scholar) with 2 cycles of slow MD annealing (MD_LEVEL = refine_4, LIBRARY_SCHEDULE = 2). An EcAdoMetDC dimer was built by superimposing the monomer homology model on the TmAdoMetDC dimer. Overall Structure—The structure of the TmAdoMetDC S63A non-processing mutant was solved by MAD phasing using Se-Met-containing crystals. The wild-type proenzyme structure was solved by molecular replacement. Both the proenzyme and the S63A mutant crystallize in the trigonal space group R3 with unit cell dimensions a = 104.9 Å and c = 69.5 Å. The asymmetric unit contains one complete dimer with overall dimensions of ∼42 Å × 33 Å × 25 Å. Crystallographic data and refinement statistics are shown in Tables II and III. A representative section of" @default.
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• W2032393676 title "Evolutionary Links as Revealed by the Structure of Thermotoga maritima S-Adenosylmethionine Decarboxylase" @default.
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