Crystal Structure of a Ternary Complex of DnrK, a Methyltransferase in Daunorubicin Biosynthesis, with Bound Products
2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês
10.1074/jbc.m407081200
ISSN1083-351X
AutoresAnna Jansson, H. Koskiniemi, Pekka Mäntsälä, Jarmo Niemi, G. Schneider,
Tópico(s)RNA modifications and cancer
ResumoOne of the final steps in the biosynthesis of the widely used anti-tumor drug daunorubicin in Streptomyces peucetius is the methylation of the 4-hydroxyl group of the tetracyclic ring system. This reaction is catalyzed by the S-adenosyl-l-methionine-dependent carminomycin 4-O-methyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-l-homocysteine and 4-methoxy-ϵ-rhodomycin T has been determined to a 2.35-Å resolution. DnrK is a homodimer, and the subunit displays the typical fold of small molecule O-methyltransferases. The structure provides insights into the recognition of the anthracycline substrate and also suggests conformational changes as part of the catalytic cycle of the enzyme. The position and orientation of the bound ligands are consistent with an SN2 mechanism of methyl transfer. Mutagenesis experiments on a putative catalytic base confirm that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This contrasts the mechanism of DnrK with that of other O-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement. One of the final steps in the biosynthesis of the widely used anti-tumor drug daunorubicin in Streptomyces peucetius is the methylation of the 4-hydroxyl group of the tetracyclic ring system. This reaction is catalyzed by the S-adenosyl-l-methionine-dependent carminomycin 4-O-methyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-l-homocysteine and 4-methoxy-ϵ-rhodomycin T has been determined to a 2.35-Å resolution. DnrK is a homodimer, and the subunit displays the typical fold of small molecule O-methyltransferases. The structure provides insights into the recognition of the anthracycline substrate and also suggests conformational changes as part of the catalytic cycle of the enzyme. The position and orientation of the bound ligands are consistent with an SN2 mechanism of methyl transfer. Mutagenesis experiments on a putative catalytic base confirm that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This contrasts the mechanism of DnrK with that of other O-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement. Daunorubicin and doxorubicin are aromatic polyketide antibiotics that exhibit high cytotoxicity and are widely applied in the chemotherapy of a variety of cancers (1Di Marco A. Gaetani M. Orezzi P. Scarpinato B.M. Silvestrini R. Soldati M. Dasdia T. Valentini L. Nature. 1964; 201: 706-707Crossref PubMed Scopus (170) Google Scholar, 2Arcamone F. Franceschi G. Penco S. Selva A. Tetrahedron Lett. 1969; 13: 1007-1010Crossref PubMed Scopus (231) Google Scholar). These and related anthracyclines consist of a cyclic polyketide backbone, 7,8,9,10-tetrahydrotetracene-5,12-quinone, glycosylated at position C7 or C10 (Fig. 1). Diversity is generated by variations in the modification of the aglycone moiety and the composition of the attached carbohydrate. Biosynthesis of daunorubicin/doxorubicin starts with the formation of the polyketide backbone catalyzed by a class II polyketide synthase with subsequent cyclization of the polyketide chain (3Shen B. Hutchinson C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6600-6604Crossref PubMed Scopus (78) Google Scholar). These steps lead to the formation of aklavinone, a common intermediate in the synthesis of most anthracyclines. This aglycone is then further modified through a series of steps, i.e. hydroxylation, glycosylation, methylester hydrolysis, decarboxylation, methylation, and, in the case of doxorubicin, oxidation by the action of tailoring enzymes (4Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mantsala P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (18) Google Scholar, 5Niemi J. Wang Y. Airas K. Ylihonko K. Hakala J. Mantsala P. Biochim. Biophys. Acta. 1999; 1430: 57-64Crossref PubMed Scopus (17) Google Scholar, 6Connors N.C. Strohl W.R. J. Gen. Microbiol. 1993; 139: 1353-1362Crossref PubMed Scopus (15) Google Scholar, 7Connors N.C. Bartel P.L. Strohl W.R. J. Gen. Microbiol. 1990; : 1887-1894Crossref Scopus (32) Google Scholar, 8Madduri K. Torti F. Colombo A.L. Hutchinson C.R. J. Bacteriol. 1993; 175: 3900-3904Crossref PubMed Google Scholar, 9Dickens M.L. Priestley N.D. Strohl W.R. J. Bacteriol. 1997; 179: 2641-2650Crossref PubMed Google Scholar). Glycosylation of these polyketide antibiotics is usually required for biological activity and often occurs as the initial modification step (3Shen B. Hutchinson C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6600-6604Crossref PubMed Scopus (78) Google Scholar, 10Matsuzawa Y. Oki T. Takeuchi T. Umezawa H. J. Antibiot. (Tokyo). 1981; 34: 1596-1607Crossref PubMed Scopus (38) Google Scholar).The genes for the entire pathway of daunorubicin biosynthesis in Streptomyces peucetius have been cloned (11Stutzman-Engwall K.J. Hutchinson C.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3135-3139Crossref PubMed Scopus (39) Google Scholar, 12Otten S.L. Stutzman-Engwall K.J. Hutchinson C.R. J. Bacteriol. 1990; 172: 3427-3434Crossref PubMed Google Scholar). The dnrK gene of this cluster codes for an anthracycline 4-O-methyltransferase that catalyzes one step in daunorubicin biosynthesis, the methylation of carminomycin at the C4 hydroxyl group (Fig. 1) (8Madduri K. Torti F. Colombo A.L. Hutchinson C.R. J. Bacteriol. 1993; 175: 3900-3904Crossref PubMed Google Scholar). The polypeptide chain of DnrK consists of 355 amino acids, and the sequence contains the DLGGGXG fingerprint that is characteristic for the binding site of S-adenosyl-l-methionine (AdoMet) 1The abbreviations used are: AdoMet, S-adenosyl-l-methionine; SAH, S-adenosyl-l-homocysteine; ϵ-T, ϵ-rhodomycinone-rhodosamine (ϵ-rhodomycin T); M-ϵ-T, 4-methoxy-ϵ-rhodomycin T; DbrT/A: 11-deoxy-β-rhodomycin T/A; AknT/A, aclacinomycin T/A; RdmB, aclacinomycin-10-hydroxylase; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviation. 1The abbreviations used are: AdoMet, S-adenosyl-l-methionine; SAH, S-adenosyl-l-homocysteine; ϵ-T, ϵ-rhodomycinone-rhodosamine (ϵ-rhodomycin T); M-ϵ-T, 4-methoxy-ϵ-rhodomycin T; DbrT/A: 11-deoxy-β-rhodomycin T/A; AknT/A, aclacinomycin T/A; RdmB, aclacinomycin-10-hydroxylase; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean square deviation. (8Madduri K. Torti F. Colombo A.L. Hutchinson C.R. J. Bacteriol. 1993; 175: 3900-3904Crossref PubMed Google Scholar). Several homologous enzymes involved in the biosynthesis of anthracyclines in various Streptomyces species have been described to date. For instance, the closest relative is DauK, which is involved in daunorubicin biosynthesis in Streptomyces sp. strain C5 (9Dickens M.L. Priestley N.D. Strohl W.R. J. Bacteriol. 1997; 179: 2641-2650Crossref PubMed Google Scholar) and displays a 95% sequence identity to DnrK. Another enzyme with high sequence similarity is RdmB (52% sequence identity), which is involved in rhodomycin biosynthesis in Streptomyces purpurascens (13Niemi J. Mäntsälä P. J. Bacteriol. 1995; 177: 2942-2945Crossref PubMed Google Scholar). The three-dimensional structure of this enzyme, determined by x-ray crystallography, shows the typical methyltransferase fold and an AdoMet binding site characteristic for these enzymes (14Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schneider G. J. Mol. Biol. 2003; 334: 269-280Crossref PubMed Scopus (31) Google Scholar). It is, however, noteworthy that RdmB does not act as a methyltransferase but is a regiospecific hydroxylase (4Wang Y. Niemi J. Airas K. Ylihonko K. Hakala J. Mantsala P. Biochim. Biophys. Acta. 2000; 1480: 191-200Crossref PubMed Scopus (18) Google Scholar) and therefore appears to be an odd member of this group of enzymes.DnrK has a rather broad substrate specificity and can methylate other anthracyclines in addition to carminomycin, for instance ϵ-rhodomycin T (Fig. 1). Here, we describe the crystal structure of a ternary complex of DnrK with the bound products SAH and 4-methoxy-ϵ-rhodomycin T (M-ϵ-T) to a 2.35-Å resolution. The structure provides insights into the recognition of the anthracycline substrate by these methyltransferases and also reveals conformational changes as part of the catalytic cycle of the enzyme. Replacement of a putative catalytic base by site-directed mutagenesis results in little change in catalytic activity. This observation suggests that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This is different from the mechanism of other related O-methyltransferases where acid/base catalysis has been demonstrated to be an essential catalytic step (15Martin J.L. McMillan F.M. Curr. Opin. Struct. Biol. 2002; 12 (Correction (2003) Curr. Opin. Struct. Biol.13, 140): 783-793Crossref PubMed Scopus (443) Google Scholar).MATERIALS AND METHODSCloning—The dnrK open reading frame was amplified from S. peucetius ATCC 27952 chromosomal DNA by PCR (using 5′-CCGAATTCCACAGCCGAACCGACGGTCG-3′ as the forward primer and 5′-CTCGAGCGGCCGCATCAGGCGCCGGTGGCC-3′ as the reverse primer), cloned in the glutathione S-transferase fusion expression plasmid pGEX4T-3 (Amersham Biosciences) using the EcoRI and NotI sites, and sequenced to confirm the published nucleotide sequence (8Madduri K. Torti F. Colombo A.L. Hutchinson C.R. J. Bacteriol. 1993; 175: 3900-3904Crossref PubMed Google Scholar).Expression and Purification—Recombinant DnrK was produced and purified as a glutathione S-transferase fusion protein according to the plasmid manufacturer's instructions (cultivation at 30 °C and induction overnight with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside). After thrombin cleavage to remove the glutathione S-transferase part, DnrK was purified by anion exchange chromatography using a HiPrep 16/10 Q XL column (0-1 m NaCl gradient in 50 mm Tris-HCl, pH 8) and gel exclusion chromatography employing a HiLoad 26/60 Superdex 200 column in an Äkta fast protein liquid chromatography system (50 mm Tris-HCl, pH 8; all components from Amersham Biosciences). The yield of pure protein was 36 mg from 5 liters of culture. The cloning procedure and the subsequent protease cleavage of the fusion protein resulted in the removal of the N-terminal methionine of DnrK and the addition of five amino acids at the N-terminal so that the N-terminal sequence of the recombinant enzyme is gspnsTAEPTV (small letters denote the amino acids resulting from the vector pGEX4T-3).Mutagenesis—PCR mutagenesis was performed by the four-primer method with the Y142W mutagenic primers 5′-GCAAGCCGTTCTggGAGGACCTGG-3′ and 5′-CCAGGTCCTCccAGAACGGCTTGC-3′ (small letters indicate the site of mutation). The original dnrK primers were used as distal primers. The PCR products were cloned, sequenced, expressed, and purified as described for the wild-type enzyme.HPLC—HPLC of anthracyclines was performed on LiChrospher 100 RP18 columns using 70:30 acetonitrile/60 mm ammonium acetate buffer, pH 3.6, as eluent. In analytical HPLC, a 5-μm particle size column (250 × 4 mm) at a flow rate of 1 ml/min was used in a Shimadzu VP series chromatography system with a diode array detector. In preparative HPLC, a 10-μm particle size column (250 × 4 mm) at a flow rate of 2.5 ml/min was employed. Liquid chromatography-mass spectrometry was performed with a PerkinElmer Life Sciences API 365 liquid chromatography-tandem mass spectrometry system (electro-spray ionization, positive ions).Anthracycline Substrates—Aclarubicin (also called aclacinomycin A or AknA) was purchased from Calbiochem, and daunorubicin was from Sigma. AknT was produced from AknA by partial hydrolysis as described (16Niemi J. Ylihonko K. Hakala J. Parssinen R. Kopio A. Mäntsälä P. Microbiology. 1994; 140: 1351-1358Crossref PubMed Scopus (50) Google Scholar) and purified by preparative HPLC. ϵ-Rhodomycin T was produced by culturing Streptomyces galilaeus ATCC 31615 transformed with the 11-hydroxylase expression plasmid pJN018 (5Niemi J. Wang Y. Airas K. Ylihonko K. Hakala J. Mantsala P. Biochim. Biophys. Acta. 1999; 1430: 57-64Crossref PubMed Scopus (17) Google Scholar) (13Niemi J. Mäntsälä P. J. Bacteriol. 1995; 177: 2942-2945Crossref PubMed Google Scholar) in E1 medium as described (16Niemi J. Ylihonko K. Hakala J. Parssinen R. Kopio A. Mäntsälä P. Microbiology. 1994; 140: 1351-1358Crossref PubMed Scopus (50) Google Scholar). After partial hydrolysis, the monoglycoside was purified by preparative HPLC. Carminomycin was produced by culturing Nonomuraea roseoviolacea subsp. carminata DSM 44170 (originally Actinomadura roseoviolacea, (17Gauze G.F. Sveshnikova M.A. Ukholina R.S. Gavrilina G.V. Filicheva V.A. Antibiotiki. 1973; 18: 675-678PubMed Google Scholar) in E1 medium. The product was recovered and purified as above. DbrA was prepared by incubating 1 mg of AknA for 2 h at 37 °C in a 10-ml reaction mixture containing 100 mm potassium phosphate buffer, pH 7, 10 μm AdoMet, 1 mm glutathione, 24 μg/ml purified aclacinomycin methylesterase (RdmC), and 37 μg/ml purified RdmB (18Wang Y. Niemi J. Mäntsälä P. FEMS Microbiol. Lett. 2002; 208: 117-122Crossref PubMed Google Scholar). The anthracycline products were recovered by solid phase extraction as described below, and DbrA was purified by preparative HPLC. DbrT was produced from DbrA by partial hydrolysis and preparative HPLC as described for AknT.Enzyme Assay—DnrK activity was assayed in a reaction mixture containing 100 mm potassium phosphate buffer, pH 7.5, 80 μm AdoMet, 40 μm substrate, and 90 μg/ml enzyme, which was incubated for 1 h at 37 °C; the reaction was terminated, and the anthracycline products were recovered by solid phase extraction using Supelco DSC-18 columns according to the manufacturer's instructions. The products were assayed by analytical HPLC.Crystallization and Data Collection—Crystallization of the ternary complex of DnrK was carried out at 20 °C using the vapor diffusion method. 2 μl of a protein solution (9.4 mg/ml DnrK, including 3 mm ϵ-rhodomycinone-rhodosamine (ϵ-T), 10 mm AdoMet, and 10 mm dithiothreitol in 50 mm Tris buffer, pH 7.5) were mixed with an equal amount of mother liquid. Hampton research kits were used for crystallization screening. Crystals were obtained after 1 week using 30% (w/v) polyethylene glycol 4000, 0.2 m sodium acetate, and 0.1 m Tris buffer (pH 8.5). One crystal form grew in space group P212121 with unit cell dimensions of a = 60.8 Å, b = 102.0 Å, and c = 124.4 Å and contained two subunits per asymmetric unit, corresponding to a solvent content of 54%. The x-ray data was collected at 100 K at beam line ID29 at the European Synchrotron Radiation Facility, Grenoble, France to a resolution of 2.5 Å. The crystals could be frozen directly due to the high content of polyethylene glycol 4000 in the mother liquid.A second crystal form of the DnrK ternary complex was obtained using 1.6 m ammonium sulfate as a precipitant in 0.1 m MES buffer at pH 6 at room temperature. Diffraction data from these crystals were collected at 100 K at beam line 7-11, MAX-lab, Lund, Sweden to 2.35 Å resolution. The crystals were of space group C2 with the cell dimensions a = 210.7 Å, b = 53.1 Å, c = 83.1Å, and β = 105°. These C2 crystals have two subunits per asymmetric unit and a solvent content of 60%.All diffraction images were indexed and processed with the program MOSFLM (19Leslie A.G.W. Joint CCP4 and ESF-EAMCB Newsletter on Protein Crystallography. 26. Daresbury Laboratory, Warrington, UK1992Google Scholar). The program SCALA of the CCP4 program package (20Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar) was used for scaling. Space group determination was based on the auto-indexing of the diffraction patterns by examining the scaling statistics and pseudo-precession photographs generated with the program PATTERN (21Lu G. J. Appl. Crystallogr. 1999; 32: 375-376Crossref Google Scholar). Details of the statistics for the diffraction data are given in Table I.Table IStatistics of data collection and structure refinementData collectionSpace groupP212121C2Mol/asuaMolecules per asymmetric unit.22Resolution (Å)2.502.35BeamlineID29BL-711Wavelength (Å)0.981.09No. observations245404293378No. unique reflections2756941714Rsym10.4 (32.2)7.1 (21.4)Completeness99.9 (99.9)94.8 (89.8)I/σ12.4 (4.5)13.9 (5.5)RefinementRwork (%)20.319.4Rfree (%)27.024.1No. amino acids (chains A/B)343/343343/343No. atomsProtein (chains A/B)2674/27712690/2690Ligands (cofactor/substrate)26/4326/43Water molecules165375B-factor from Wilson plot (Å2)3129B-factor (Å2)Chain A protein29.126.7Chain B protein27.331.9Chain A ligands (cofactor/substrate)22.2/26.426.9/29.1Chain B ligands (cofactor/substrate)20.7/70.428.3/41.1Waters22.335.1R.m.s.d from ideal geometryBond length (Å)0.0130.011Bond angles (°)1.5311.423Figure of merit0.7890.839Ramachandran plot (%)Residues in most favored regions92.793.3Residues in additional allowed7.36.2Residues in generously allowed00.2a Molecules per asymmetric unit. Open table in a new tab Structure Determination and Refinement—The structure of the DnrK ternary complex was solved initially in the P212121 crystal form by molecular replacement using the program Molrep (22Vagin A.A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4120) Google Scholar). The search model was that of a ternary complex of the homologous hydroxylase RdmB, AdoMet, and substrate. 2A. Jansson, G. Schneider, and J. Niemi, manuscript in preparation. The monomer of RdmB was used as a search model, and a rotation and translation search was used to find the position of the two subunits in the asymmetric unit, giving a solution with an R-factor of 57.5% and correlation coefficient of 0.39.The program REFMAC 5.0 (23Murshudov G. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-253Crossref PubMed Scopus (13776) Google Scholar) was employed for refinement by the maximum likelihood residual. A set of reflections, ∼8% for every data set, was set aside for the calculation of Rfree. The protocol consisted of initial rigid body refinement, which was followed by restrained conjugate gradient minimization. Isotropic B-factor refinement and bulk solvent correction were used. Non-crystallographic symmetry restraints were applied whenever possible to improve the data to parameter ratio, but care was taken not to include parts of the structures that differed between the two non-crystallographic symmetry-related subunits. Thus, the rigid Rossman-like fold was tightly restrained, whereas other parts of the structure were kept rather loosely restrained by noncrystallographic symmetry. Water molecules were added by ARP/WARP (24Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2562) Google Scholar). Rounds of refinement were followed by manual inspection of the electron density maps using the program O (25Jones T.A. Zou J. Cowan S. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). The refinement protocol converged at an R-factor of 20.3% and an Rfree of 27.0%.This refined model was used to solve the C2 crystal form by molecular replacement using Molrep (22Vagin A.A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4120) Google Scholar). A solution was found using the monomer as a search model with a correlation coefficient of 0.266 and an R-factor of 56.2%. The bound ligands had been removed from the search model, and the correctness of the solution was verified by electron density appearing for these ligands in the initial 2Fo - Fc and Fo - Fc electron density maps. Refinement of this crystal form followed the same procedure as described above, and the final model has an R-factor of 19.4% and an Rfree of 24.1%.The geometry of the structures was analyzed with PROCHECK (26Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J. J. Appl. Crystallogr. 1993; 26: 282-291Crossref Google Scholar), and the details of the refinement statistics are summarized in Table I. The refined atomic coordinates and observed structure factors have been deposited in the Protein Data Bank with the accession numbers 1TW2 (DnrK/SAH/M-ϵ-T, P212121) and 1TW3 (DnrK/SAH/M-ϵ-T, C2).Structural Comparisons—Pairwise and multiple sequence alignments were made using BLAST (27Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar) and ClustalW (28Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55190) Google Scholar), respectively. Secondary structure alignments were carried out with TOP (29Lu G. J. Appl. Crystallogr. 2000; 33: 176-183Crossref Scopus (193) Google Scholar) and the LSQ option in O, using default parameters (25Jones T.A. Zou J. Cowan S. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). Figures were drawn using PyMOL (30DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlo, CA2002Google Scholar), VMD (31Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14 (27-38): 33-38Crossref PubMed Scopus (36841) Google Scholar), and BOBSCRIPT (32Esnouf R.M. J. Mol. Graph. Model. 1997; 15: 133-138Crossref Scopus (1794) Google Scholar).RESULTSIn Vitro Methylation of Anthracyclines by DnrK—Purified DnrK was tested for methyl transferase activity in vitro using different anthracycline glycosides. Carminomycin, ϵ-T, DbrT, and AknT produced new HPLC peaks. The product from carminomycin methylation co-eluted with authentic daunorubicin, and the product from ϵ-T methylation had a mass spectrum consistent with O-methylation. The identity of this product as the 4-O methylated compound was later verified by crystallographic analysis (see below). On the other hand, no new products were observed for AknA or DbrA, indicating that DnrK cannot methylate substrates with a tri-sugar chain.Activity of the Y142W Mutant—When ϵ-T was incubated in the presence of equal amounts of recombinant native DnrK and the purified mutant Y142W, methylation by the mutant enzyme proceeded at 48% of the rate of the recombinant native control as assessed by the HPLC peak area.Quality of the Maps and the Model—DnrK was co-crystallized with the substrate ϵ-T and the cofactor AdoMet. Two crystal forms, P212121 and C2, were obtained under different crystallization conditions, and these crystals diffracted to 2.5- and 2.35-Å resolution, respectively. The structures of both of the complexes were determined with molecular replacement. The final P212121 model is a dimer consisting of amino acids 14-351 in chain A and 3-352 in chain B, two SAH molecules, two product molecules, and 165 water molecules. The only missing residues are flexible residues at the N- and C termini of the protein. The electron density for the polypeptide chain and the bound ligands is well defined (Fig. 2). The A and B chains superpose with an r.m.s.d. of 1.28 Å for all Cα atoms. This relatively high r.m.s.d. value is due to localized differences in the conformation of the polypeptide chain described further below.Fig. 2Electron density maps.Top, stereo picture of the initial, unrefined 2Fo - Fc electron density map for the bound product and surrounding amino acids in the active site of DnrK. The map is contoured at 1 σ. The final refined model of the enzyme-SAH-M-ϵ-T complex is superimposed. Bottom, stereo picture of a difference Fo - Fc electron density map obtained after modeling the complex as the enzyme-AdoMet-substrate complex. Positive contours are shown in red, and negative contours are in green. The negative difference electron density at the position of the methyl group of AdoMet and the positive difference electron density adjacent to the 4-hydroxyl oxygen atom of ϵ-T shows that methyl group transfer has occurred during crystallization The map is contoured at 3 σ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The model for DnrK in space group C2 comprises amino acids 12-351 in chain A and 12-351 in chain B, two SAH molecules, two product molecules, and 375 water molecules. As in the case of the complex crystallized in space group P212121, the electron density for the polypeptide chain and the bound ligands is well defined, except for a few residues at the N- and C termini. Residue Glu-283 in the C2 model is the only amino acid in the generously allowed region of the Ramachandran plot, but it is well defined by electron density. The two chains in space group C2 give a r.m.s.d. value of 0.57 Å upon the superposition of all Cα atoms.The structures of the subunits determined in the two different space groups do not differ significantly from each other, with one exception (see below). The C2 and P212121 dimers superimpose with an r.m.s.d. difference of 0.47 Å for the A chains and 1.35 Å for the B chains. Overall, in both crystal forms the A chain is better defined in density than the B chain, including the bound ligands. This is also reflected by the B-factors for the bound ligands, which are consistently higher for the B than for the A chains (Table I). In the following we will mainly discuss the model of the DnrK ternary complex determined in the C2 crystal form because of its higher resolution and better electron density maps.Overview of the Structure—The subunit of DnrK is built up by an N-terminal domain with a mainly helical structure except for two β-strands, a middle all-helical domain, and the C-terminal Rossman-like fold comprising a central parallel β-sheet (β3-β9) surrounded by eight helices (γ13-α21). (Fig. 3). The C-terminal domain contains the binding site for the cofactor AdoMet with the conserved DLGGGXG fingerprint. The substrate/product is positioned between the middle and C-terminal domains (Fig. 3), and residues from both of these domains are involved in binding the substrate. The fold of DnrK very much resembles that of RdmB (14Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schneider G. J. Mol. Biol. 2003; 334: 269-280Crossref PubMed Scopus (31) Google Scholar), the AdoMet-dependent hydroxylase homologous to DnrK. Both subunits superimpose with an r.m.s.d. difference of 1.14 Å for 345 Cα atoms. The only difference in the secondary structure elements between the two enzymes is an additional helix (γ19) in DnrK, which is inserted between β7 and α20.Fig. 3Schematic view of the homodimer of the ternary complex of DnrK-SAH-product. Monomer A is yellow, monomer B is purple, and the bound ligands are shown in cyan ball-and-stick models and labeled. Secondary structure elements in the A monomer are labeled.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The N-terminal domain is extensively involved in the dimer interface (buried surface area of 3697 Å2) that involves 15 hydrogen bonds but is otherwise dominated by hydrophobic interactions. The secondary elements mainly engaged in dimer formation are α1, α2, γ6, α7, α8, and α19. In both crystal forms DnrK is found as a tightly packed dimer. Analysis of crystal packing shows that the interactions with neighboring molecules in the crystal lattice are rather weak in space group in P212121 (pH 8.5). The largest interaction area with an adjacent molecule is only 511 Å2, indicative of crystal contacts rather than a protein-protein interface. In space group C2, the interface to the closest neighbor comprises 930 Å2 and might thus indicate formation of a tetramer. The relatively small size of this interface suggests that the tetramer may not be very stable. Gel filtration experiments with DnrK, both in the absence and presence of AdoMet and substrate, indicate that at low (6.5) and high pH (7.5) the enzyme forms a mixture of dimer and tetramers in solution, consistent with the crystallographic analysis.AdoMet/SAH Binding Site—In DnrK the cofactor AdoMet (with the exception of the methyl group) is well defined by electron density. The AdoMet/SAH binding site is located in the C-terminal domain at the carboxyl end of the β-strands of the nucleotide binding fold, and the cofactor is bound to DnrK in a similar manner as in other small molecule methyltransferases. The cofactor interacts with the enzyme via an extensive hydrogen bond network and a few hydrophobic interactions (Fig. 4, top). The adenine ring forms stacking interactions with the side chains of Trp-257 and Phe-237, and Asp-236 forms a hydrogen bond to the N6 amino group. The ribose moiety is anchored to DnrK through hydrogen bonds of the O2* and O3* hydroxyl groups to the side chains of Glu-209 and Arg-152. Ser-251 interacts with both the carboxyl group as well as the amino group of AdoMet/SAH, and the latter also forms a hydrogen bond to the main chain carbonyl oxygen of Gly-186. Most of these interactions are conserved between DnrK and RdmB with the exception of Arg-152, which is replaced by an alanine residue in RdmB. Furthermore, the hydrogen bond of the carboxyl group of AdoMet-SAH to the side chain of Tyr-171 (corresponding to Phe-167 in DnrK) observed in RdmB (14Jansson A. Niemi J. Lindqvist Y. Mäntsälä P. Schne
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