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• W2088998457 abstract "Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH4) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH4 product, l-threo-BH4 (6R-(1′S,2′S)-5,6,7,8-BH4), whereas animal enzymes produce l-erythro-BH4 (6R-(1′R,2′S)-5,6,7,8-BH4) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 Å resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180° around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold. Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH4) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH4 product, l-threo-BH4 (6R-(1′S,2′S)-5,6,7,8-BH4), whereas animal enzymes produce l-erythro-BH4 (6R-(1′R,2′S)-5,6,7,8-BH4) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 Å resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180° around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold. Sepiapterin reductase (SR) 2The abbreviations used are: SR, sepiapterin reductase; cSR, C. tepidum SR; mSR, mouse SR; hSR, human SR; BH4, tetrahydrobiopterin; PDB, Protein Data Bank; PPH4, 6-pyruvoyltetrahydropterin; SeMet, selenomethionine. 2The abbreviations used are: SR, sepiapterin reductase; cSR, C. tepidum SR; mSR, mouse SR; hSR, human SR; BH4, tetrahydrobiopterin; PDB, Protein Data Bank; PPH4, 6-pyruvoyltetrahydropterin; SeMet, selenomethionine.; EC 1.1.1.153) catalyzes the last step of the de novo synthesis of tetrahydrobiopterin (BH4) from GTP. BH4 is a well known essential cofactor for aromatic amino acid hydroxylases (1Kaufman S. Annu. Rev. Nutr. 1993; 13: 261-286Crossref PubMed Scopus (149) Google Scholar) and nitric-oxide synthase (2Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (808) Google Scholar) in humans and other higher organisms (3Thöny B. Auerbach G. Blau N. Biochem. J. 2000; 347: 1-16Crossref PubMed Scopus (711) Google Scholar, 4Werner-Felmayer G. Golderer G. Werner E.R. Curr. Drug Metab. 2002; 3: 159-173Crossref PubMed Scopus (140) Google Scholar). BH4 deficiency in human results in severe neurological disorders like atypical phenylketonuria and monoamine neurotransmitter deficiency and is also implicated in Parkinson disease, Alzheimer disease, and depression (3Thöny B. Auerbach G. Blau N. Biochem. J. 2000; 347: 1-16Crossref PubMed Scopus (711) Google Scholar, 5Blau N. Bonafe L. Thöny B. Mol. Genet. Metab. 2001; 74: 172-185Crossref PubMed Scopus (151) Google Scholar).The pathway of the de novo biosynthesis of BH4 from GTP involves only three enzymes, GTP cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyltetrahydropterin (PPH4) synthase (EC 4.2.3.12), and SR. SR catalyzes NADPH-dependent reduction of the di-keto group in the C6 side chain of PPH4 to BH4 (6Katoh S. Sueoka T. J. Biochem. (Tokyo). 1987; 101: 275-278Crossref PubMed Scopus (22) Google Scholar) (Fig. 1). Biochemical and crystallographic analyses of murine SR suggest that SR has both reductase and isomerase activities; SR reduces first the C1′ carbonyl group and subsequently catalyzes an isomerization reaction shifting the C2′ carbonyl group to the C1′ position and then the second reduction of the carbonyl group to produce l-erythro-BH4 (7Katoh S. Sueoka T. J. Biochem. (Tokyo). 1988; 103: 286-289Crossref PubMed Scopus (16) Google Scholar, 8Auerbach G. Herrmann A. Gutlich M. Fischer M. Jacob U. Bacher A. Huber R. EMBO J. 1997; 16: 7219-7230Crossref PubMed Scopus (75) Google Scholar). Although SR is the only enzyme completing the step alone, other enzymes such as aldose reductase or carbonyl reductase also participate in the terminal step of BH4 synthesis (9Milstien S. Kaufman S. Biochem. Biophys. Res. Commun. 1989; 165: 845-850Crossref PubMed Scopus (36) Google Scholar, 10Park Y.S. Heizmann C.W. Wermuth B. Levine R.A. Steinerstauch P. Guzman J. Blau N. Biochem. Biophys. Res. Commun. 1991; 175: 738-744Crossref PubMed Scopus (71) Google Scholar). Aldose reductase catalyzes C2′-specific reduction of PPH4 to produce 1′-oxo-2′-hydroxytetrahydropterin, which is then oxidized nonenzymatically to sepiapterin (6-lactoyl-dihydropterin) (10Park Y.S. Heizmann C.W. Wermuth B. Levine R.A. Steinerstauch P. Guzman J. Blau N. Biochem. Biophys. Res. Commun. 1991; 175: 738-744Crossref PubMed Scopus (71) Google Scholar, 11Bonafe L. Thony B. Penzien J.M. Czarnecki B. Blau N. Am. J. Hum. Genet. 2001; 69: 269-277Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Sepiapterin is catalyzed in vivo by SR to 7,8-dihydrobiopterin, which is further reduced to BH4 by dihydrofolate reductase, constituting an alternative pathway of BH4 synthesis (10Park Y.S. Heizmann C.W. Wermuth B. Levine R.A. Steinerstauch P. Guzman J. Blau N. Biochem. Biophys. Res. Commun. 1991; 175: 738-744Crossref PubMed Scopus (71) Google Scholar, 11Bonafe L. Thony B. Penzien J.M. Czarnecki B. Blau N. Am. J. Hum. Genet. 2001; 69: 269-277Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar).Although l-erythro-BH4 (6R-(1′R,2′S)-5,6,7,8-BH4) is common in nature, other stereoisomers such as d-threo-BH4 (6R-(1′R,2′R)) and l-threo-BH4 (6R-(1′S,2′S)) are also found (12Klein R. Thiery R. Tatischeff I. Eur. J. Biochem. 1990; 187: 665-669Crossref PubMed Scopus (42) Google Scholar). d-threo-Form (dictyopterin) was isolated from Dictyostelium (12Klein R. Thiery R. Tatischeff I. Eur. J. Biochem. 1990; 187: 665-669Crossref PubMed Scopus (42) Google Scholar), and l-threo stereoisomer was isolated from Chlorobium tepidum as a glycoside (tepidopterin, l-threo-BH4-N-acetylglucosamine) (13Cho S.H. Na J.U. Youn H. Hwang C.S. Lee C.H. Kang S.O. Biochim. Biophys. Acta. 1998; 1379: 53-60Crossref PubMed Scopus (43) Google Scholar). It was shown that C. tepidum SR (cSR) catalyzes the reduction of not only sepiapterin to l-threo-H2-biopterin (14Cho S.H. Na J.U. Youn H. Hwang C.S. Lee C.H. Kang S.O. Biochem. J. 1999; 340: 497-503Crossref PubMed Scopus (13) Google Scholar) but also PPH4 to l-threo-BH4 (15Choi Y.K. Jun S.R. Cha E.Y. Park J.S. Park Y.S. FEMS Microbiol. Lett. 2005; 242: 95-99Crossref PubMed Scopus (7) Google Scholar). In contrast, d-threo-BH4 was not produced by SR alone but by a collaborative work of SR and aldose reductase-like protein in Dictyostelium (16Kim Y.A. Chung H.J. Kim Y.J. Choi Y.K. Hwang Y.K. Lee S.W. Park Y.S. Mol. Cell. 2000; 10: 405-410Google Scholar, 17Choi Y.K. Park J.S. Kong J.S. Morio T. Park Y.S. FEBS Lett. 2005; 579: 3085-3089Crossref PubMed Scopus (10) Google Scholar).cSR has high protein sequence similarities (∼43%) with mouse SR (mSR) (PDB code 1SEP) and human SR (hSR) 3E. Ugochukwa, K. Kavanagh, S. Ng, C. Arrowsmith, A. Edward, M. Sundstrom, F. von Delf, and U. Oppermann, unpublished material. 3E. Ugochukwa, K. Kavanagh, S. Ng, C. Arrowsmith, A. Edward, M. Sundstrom, F. von Delf, and U. Oppermann, unpublished material. (PDB code 1Z6Z) and is predicted to share the same fold as the homologous mSR (8Auerbach G. Herrmann A. Gutlich M. Fischer M. Jacob U. Bacher A. Huber R. EMBO J. 1997; 16: 7219-7230Crossref PubMed Scopus (75) Google Scholar) (Fig. 2). It was, therefore, a question how the structurally similar enzymes catalyze the synthesis of different isomeric forms of BH4. Thus, we have determined the crystal structures of cSR alone and in complex with NADP and sepiapterin at 2.1 and 1.7 Å of resolution, respectively. The overall structure of cSR is quite similar to that of mSR. However, significant structural differences are observed in the substrate binding site of the two enzymes. Here, we report a novel ligand binding mode that determines the stereospecific catalysis of cSR in the production of l-threo-BH4.FIGURE 2Sequence alignment of SRs from Chlorobium and mammals. The amino acid sequence alignment of SRs from C. tepidum, Dictyostelium discoideum, human, rat, mouse, Takifugu rubripes, and Drosophila melanogaster is shown. A column is framed in red when residues are identical and boxed when at least four of its residues (red) are homologues. Residues for cofactor binding are marked with a plus (+), central residues for substrate binding in the Chlorobium are marked with an asterisk (*), and catalytic triad residues are marked with a number sign (#). Correspondence between amino acid sequences and the secondary structure defined by DSSP (38Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12100) Google Scholar) is given on top for Chlorobium SR; helices are shown as coils, and β-strands are shown as arrows. The alignment was done by ClustalX (39Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35195) Google Scholar), and the figure was drawn with ESPript (27Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2505) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)EXPERIMENTAL PROCEDURESCloning, Expression, and Purification—The cSR was cloned, expressed, and purified following the protocols described previously (18Supangat S. Choi Y.K. Young S.P. Son D. Han C.D. Lee K.H. Acta Crystallogr. F. 2005; 61: 202-204Crossref Scopus (2) Google Scholar). Briefly, cSRs were cloned into pET-28b and transformed into Escherichia coli strain BL21(DE3) for free cSR and B834(DE3)PLysS for selenomethionine (SeMet) derivative, respectively. The soluble protein was purified by nickel-agarose affinity, anion exchange, and gel filtration chromatography. The protein was concentrated to 10 mg/ml in 20 mm Tris-HCl, pH 8.0.Crystallization and Data Collection—Free cSR and the SeMet-derivative proteins of cSR were crystallized following the protocols described previously (18Supangat S. Choi Y.K. Young S.P. Son D. Han C.D. Lee K.H. Acta Crystallogr. F. 2005; 61: 202-204Crossref Scopus (2) Google Scholar). To prepare crystals of cSR-NADP-sepiapterin complex, protein was mixed with NADP and sepiapterin to final concentrations of 10 mm and incubated for 3 h before set-up. Crystals of free, SeMet-derivative protein and cSR-NADP-sepiapterin complex were obtained by hanging drop vapor diffusion method at 18 °C in a drop containing 4 μl of protein solution and 1 μl of a mixture of 4 μl of reservoir solution (0.2 m MgCl2, 0.1 m Tris-HCl, pH 8.5, 34% polyethylene glycol 400) and 1 μl of additive (1 m guanidine hydrochloride).X-ray diffraction data for free cSR and cSR-NADP-sepiapterin complex were collected from a single crystal to 2.1- and 1.7-Å resolution using x-ray of wavelength 1.1273 Å and a Bruker CCD detector at station 6B of the Pohang Accelerator Laboratory, Pohang, Republic of Korea, respectively. Diffraction data for SeMet derivative was collected from a single crystal to 2.1 Å of resolution at station 6A at the Photon Factory at the High Energy Accelerator Research Organization, Tsukuba, Japan. All diffraction images were indexed, integrated, and scaled using the HKL2000 suite (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar). Both free and SeMet-derivative crystals are in space group R32, whereas the cSR-NADP-sepiapterin complex is in space group P212121. Data collection statistics are presented in Table 1.TABLE 1Crystallographic statisticsNativeSeMetNADP-Sepiapterin complexData statisticsSpace groupR32aIIexagonal indexing with α = β = 90° and γ = 120°R32P212121Unit cell (Å)a = 202.14, c = 210.02a = 201.14, c = 210.18a = 84.36, b = 97.48, c = 123.24No. of chains in AU4 molecules4 molecules4 moleculesPeakEdgeRemoteWavelength (Å)1.1230.9780.97930.97041.123Resolution (Å)50 = 2.15 (2.23–2.15)bNumbers in parentheses represent values in the highest resolution shell25 = 2.1 (2.18 = 2.1)35 = 1.7 (1.76 = 1.7)Completeness (%)99.2 (98.5)99.999.9 (99.7)99.999.8 (93.8)(99.7)(99.7)Rsym(I)cRsym(I) = (Σhkl|I–〈I〉| /Σhkl〈I〉), where hkl are independent Miller indices (%)14.6dThis value is a little higher because of high mosaicity of the data (89.7)11.310.4 (64)10.97.4 (51.9)(60.2)(66.7)Redundancy1910.210.310.17〈I/σ(I)〉7.011.111.011.619.3Figure of merit0.47/0.68eAs indicated by SOLVE/RESOLVERefinementProtein atoms73867955Water molecules284369RfrecfRfree = Σhkl|Fo–Fc|/Σhkl, Fo, where hkl is a free set; Rwork = Σhkl|Fo–Fc|/ΣhklFo, where hkl is a working set (%)23.8 (30.17)21.3 (28.08)RworkfRfree = Σhkl|Fo–Fc|/Σhkl, Fo, where hkl is a free set; Rwork = Σhkl|Fo–Fc|/ΣhklFo, where hkl is a working set (%)21.9 (28.31)20.0 (25.90)Root mean square deviationsBond length (Å)0.0070.005Bond angle (°)1.261.29Mean B-factors (Å2)Protein3324Water3328NADP22Sepiapterin47a IIexagonal indexing with α = β = 90° and γ = 120°b Numbers in parentheses represent values in the highest resolution shellc Rsym(I) = (Σhkl|I–〈I〉| /Σhkl〈I〉), where hkl are independent Miller indicesd This value is a little higher because of high mosaicity of the datae As indicated by SOLVE/RESOLVEf Rfree = Σhkl|Fo–Fc|/Σhkl, Fo, where hkl is a free set; Rwork = Σhkl|Fo–Fc|/ΣhklFo, where hkl is a working set Open table in a new tab Structure Determination and Refinement—The structure of cSR was determined by a multiple wavelength anomalous diffraction experiment using the SeMet derivative (20Hendrickson W.A. Science. 1991; 254: 51-58Crossref PubMed Scopus (1011) Google Scholar). Attempts to solve the structure by molecular replacement using the program AMoRe (21CCP4Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar) with the mSR as a search model have given no useful solutions. Diffraction was recorded at peak, inflection, and high energy remote points for multiple wave-length anomalous diffraction phasing. 33 selenium sites were located, and phases were calculated using SOLVE/RESOLVE (22Terwilliger T.C. Berendzen J. Acta Crystallogr. D. 1999; 55: 1872-1877Crossref PubMed Scopus (64) Google Scholar). The atomic model was built into the electron density with the program O (23Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). The structure refinement was carried out with the program CNS (24Brünger 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. D. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar). The stereochemistry of the structure was checked by PROCHECK (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). For free cSR, in the Ramachandran plot, 88.3% of all the residues are in most favored region, and for cSR-NADP-sepiapterin complex, 92.1% of all the residues are in most favored region. Figures were prepared using PyMOL (26DeLano W.L. The pyMol Molecular Graphics System. DeLano Scientific, San Carlos, CA2001Google Scholar). The statistics of the structure refinement are presented in Table 1.RESULTS AND DISCUSSIONStructure Determination—The crystal structure of the free enzyme of cSR was solved at 2.1 Å of resolution using the selenomethionyl multiple wavelength anomalous diffraction method. There are four molecules in the asymmetric unit of the crystal, and 33 of the expected 40 selenium positions were located from the anomalous difference data. The current atomic model has an R value of 20%, and most of the residues (91%) are in the most favored region of the Ramachandran plot (data not shown). The crystallographic data and refinement statistics are summarized in Table 1.The structure of cSR in complex with NADP and sepiapterin has been determined at 1.7 Å of resolution (Table 1) from a co-crystal obtained from the mixture of protein, NADP, and sepiapterin. The complex was packed in space group P212121 different from the free enzyme in space group R32. There are four molecules in the asymmetric unit and one NADP per molecule and one sepiapterin molecule modeled in the tetramer. The other three sepiapterin molecules have weak electron densities and are not included in the model. In both free enzyme and NADP-sepiapterin complex, the N-terminal hexahistidine tag and residue 1 are disordered. In addition, residues 194–203 of the free enzyme are disordered and not included in the atomic model. Also, residues 198–202 in the complex have weak electron densities for side chains and are modeled as alanines.Overall Structure of cSR—In the cSR crystal structure, each monomer (244 residues) folds into a single domain α/β-structure. A seven-stranded parallel β-sheet in the center of the molecule is sandwiched by two layers of three α-helices (αA, αB, αF and αC, αD, αE), homologous to those found in other short chain dehydrogenase/reductase including mSR and hSR (8Auerbach G. Herrmann A. Gutlich M. Fischer M. Jacob U. Bacher A. Huber R. EMBO J. 1997; 16: 7219-7230Crossref PubMed Scopus (75) Google Scholar, 28Ghosh D. Wawrzak Z. Weeks C.M. Duax W.L. Erman M. Structure. 1994; 2: 629-640Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 29Rossmann M.G. Liljas A. Bränden C.-I. Banaszak L.J. The Enzymes. Academic Press, Inc., New York1975: 61-102Google Scholar)3 (Fig. 3A). The central six strands form a typical dinucleotide binding motif (29Rossmann M.G. Liljas A. Bränden C.-I. Banaszak L.J. The Enzymes. Academic Press, Inc., New York1975: 61-102Google Scholar) composed of βαβ units. A Dali (30Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1276) Google Scholar) search for structurally similar proteins showed that the cSR chain topology matches most closely other short chain dehydrogenase/reductases (PDB codes 1YBV, 1FDS, 1CYD, 1GZ6, 1E6W, 1OAA, 1BDB, 1E7W) with Z scores greater than 24. The overall folding of cSR is similar to mSR and hSR, sharing a common tertiary structure with an NADP(H) cofactor binding site and a conserved YXXXLYS sequence motif with root mean square deviations of 0.90 and 1.16 Å to hSR and mSR among Cα atoms in the central β-sheet, respectively (calculated with lsqkab in CCP4 Suite) (21CCP4Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar).FIGURE 3Overall structure of cSR. A, stereoview of ribbon representation of the cSR monomer. β-Strands and α-helices are labeled in alphabetical order from the N terminus. NADP (gray, carbon; red, oxygen; blue, nitrogen; orange, phosphate) and sepiapterin (yellow, carbon; red, oxygen; blue, nitrogen) are shown as sticks. B, ribbon representation of a cSR tetramer formed by two dimers in the asymmetric unit (chain A is in orange, B is in green, C is in cyan, and D is in magenta). There are two different dimer interfaces, one between the orange and green monomers and the other between the orange and cyan monomers. C, superposition of the SR monomers from Chlorobium (magenta), mouse (green; PDB code 1SEP) and human (cyan; PDB code 1Z6Z) with NADP (magenta) and sepiapterin (magenta) bound in the cSR. The largest differences from the three structures are marked with arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)However, significant differences between cSR and mSR or hSR molecules were observed in the substrate binding region around the active site. The loop between βF and αF (residues Val-191 to Met-207; 17 amino acids) in cSR is much shorter than that in mSR (residues Leu-202 to Leu-232; 31 amino acids) and in hSR (residues Leu-198 to Leu-228; 30 amino acids) (Fig. 3C). Also, the residues in the loop between βF and αF show low sequence similarities among these enzymes. The residues at the C-terminal region from Arg-235 to Ile-241 in the cSR extend longer so that it covers up the side of the cavity that is open in the mSR and hSR (Fig. 3C). Therefore, the short loop and the long C-terminal extension in the cSR make changes in the shape and the size of the substrate binding cavity. These changes around the binding cavity may explain the different stereospecific catalysis of the enzyme.Structure of the cSR Tetramer—cSR is a tetramer in the asymmetric unit of the crystal formed by two homodimers, although cSR exists as a homodimer in solution, confirmed by gel filtration and cross-linking experiment (data not shown). The four cSR molecules in the asymmetric unit have essentially the same conformation, with average root mean square distances of 0.21 Å between equivalent Cα atoms for the free enzyme and 0.27 Å for the NADP-sepiapterin complex (calculated with lsqkab for all Cα pairs). In the tetramer there are two different monomer-monomer interactions by the associations of two cSR monomers, each monomer contacting with two neighboring monomers in a head-to-tail fashion related by a noncrystallographic 2-fold axis (Fig. 3B). The interface between chain A and B is formed by a four-helix bundle consisting of helices αD and αE of each chain. A number of hydrophobic interactions are found in this interface area, involving Phe-110, Phe-122, Phe-123, and Phe-163 from each monomer together with a salt bridge between Asp-111 of monomer A and Lys-119 of monomer B. Another dimer interface is formed between chain A and C by contacts between strand βG and helix αF from each monomer. In this dimer the β-sheet of each monomer is connected by strand βG in an antiparallel manner to each other, forming a central 14-stranded β-sheet. There is an additional salt bridge at the interface formed between Asp-212 and Arg-226 of helix αF from each monomer. No interactions are found between chain A and D, leaving a hole in the center of the tetramer filled with many solvent molecules.Active Site in cSR—In the cSR-NADP-sepiapterin complex structure, the active site can be identified from the positions of the bound NADP and sepiapterin (Fig. 4B). The pocket for substrate is located around at the C terminus formed by the three long loops between strand βD and helix αD, between βE and αE, and between βF and αF and also by a C-terminal extension of strand βG. The loop between βF and αF has two additional short helices αFG1 and αFG2 that are partially disordered in the absence of substrate (Fig. 3A). The substrate binding site is at the C-terminal end of the β-strands above the nicotinamide ring of the NADP. The active site cavity in the NADP-sepiapterin complex is formed by hydrophobic and polar residues Val-146, Ala-147, Phe-99, Arg-150, Phe-152, Ser-145, Tyr-158, Met-195, Arg-235, Asp-240, and Ile-241.FIGURE 4The sepiapterin binding mode. A, stereoview of the electron density of the sepiapterin, NADP, and Met-195. The electron density map (2Fo–Fc) contoured in 1σ of the sepiapterin (yellow, carbon; red, oxygen; blue, nitrogen), NADP (gray, carbon; red, oxygen; blue, nitrogen; brown, phosphate), and Met-195 (green, carbon; red, oxygen; blue, nitrogen; orange, sulfur) is shown in blue. B, stereographic diagram of the active site in the cSR complex. Sepiapterin, residues Phe-99, Ser-145, Ser155, Tyr-158, Lys-162, Met195, Trp-196, Asp-240, and Ile-241, and NADP are shown as sticks. Water (Wat) molecules are shown as spheres (red). Hydrogen bonds between sepiapterin and the residues are shown in dashed lines (red). C, ribbon diagram of superposition of the cSR (pale magenta) and mSR (pale cyan) at the active site. The sepiapterin in cSR and the sepiapterin in mSR (cyan, carbon; red, oxygen; blue, nitrogen) are shown as sticks. The central residues for substrate binding and catalysis are shown as sticks; that is, residues Phe-99, Tyr-158, Ser-145, and Trp-196 from cSR and residues Tyr-171 and Ser-158 from mSR (cyan, carbon; red, oxygen; blue, nitrogen). The shifts of loops in cSR from the positions in mSR are marked as arrows in red. Because of these shifts, sepiapterin in cSR cannot bind to the site in which sepiapterin in mSR is bound. D, stereographic diagram of substrate binding mode. Rotation of the sepiapterin is represented by the rotation axis, and the direction of the rotation is marked as an arrow in black. Sepiapterin in cSR, sepiapterin in mSR (white blue), NADP, and residues are shown as sticks.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Enzymes of the short chain dehydrogenase/reductase family contain a highly conserved Ser-Tyr-Lys triad at the active site (32Jörnvall H. Persson B. Krook M. Atrian S. Gonzalez-Duarte R. Jeffery J. Ghosh D. Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1152) Google Scholar). In the cSR crystal structures three residues forming a Ser-Tyr-Lys triad at the active site are well conserved as in other SRs (Fig. 4C). The tyrosine residue Tyr-158 is oriented for optimal hydride transfer from NADPH to the carbonyl functional group of the sepiapterin and plays a central role in the active site for the catalysis. The basic residue Lys-162, positioned close to Tyr-158, may stabilize the resulting tyrosinate. The Ser-145, which is located in hydrogen bond distance to the C1′-side chain oxygen, is also involved in proton transfer and stabilization for the carbonyl group (33Fujimoto K. Hara M. Yamada H. Sakurai M. Inaba A. Tomomura A. Katoh S. Chem. Biol. Interact. 2001; 130: 825-832Crossref PubMed Scopus (18) Google Scholar) (Fig. 4C).The substrate acceptor site in the cSR structure is different from that found in mSR (Fig. 4C). In the mSR-NADP-sepiapterin complex structure, the substrate binding site is located at the end of strands βD and βE between residues Leu-159 and Pro-199. However, the site is not accessible any more in the cSR structure for the substrate because it is occupied and blocked by the side chains of Val-146 and Arg-235. In addition, the strand βE shifted closer by 3.1 Å toward the βD strand from the position in the mSR structure, which leaves no space enough for the substrate binding around this region (Fig. 4C). As a result, the substrate binding site in the cSR has a 10-Å deep pocket shallower than a 15-Å deep pocket in the mSR and is open toward the C-terminal bottom of the molecule.Conformational changes by substrate binding occur in the cSR structure as seen in a substrate binding loop in the structure of 7α-HSDH (31Tanaka N. Nonaka T. Nakanishi M. Deyashiki Y. Hara A. Mitsui Y. Structure. 1996; 4: 33-45Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), although no changes were observed in the mSR structure. Without the substrate binding, the loop region of residues Val-191 to Met-207 in helices αFG1 and αFG2 is not defined well and has high temperature factors. Upon substrate binding, the side chains of residues 193–197 have clear electron densities, and the side chain of Trp-196 in the loop specifically interacts with the pterin moiety of the substrate (Fig. 3C).NADP Binding Mode—In the cSR complex the cofactor NADP is well ordered and has well defined electron density. The bound NADP molecule has an extended conformation with the adenine ring in anti and the nicotinamide ring in syn conformations and binds at the C-terminal side of the parallel β-sheet in a similar way as seen in mSR (8Auerbach G. Herrmann A. Gutlich M. Fischer M. Jacob U. Bacher A. Huber R. EMBO J. 1997; 16: 7219-7230Crossref PubMed Scopus (75) Google Scholar). Both the ribose rings in the NADP have C2-endo puckering conformations. Most of the residues in direct contact with the NADP in the cSR complex structure ar" @default.
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• W2088998457 title "Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin" @default.
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