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• W2061319036 abstract "The crystal structure of the nitroreductase enzyme from Enterobacter cloacae has been determined for the oxidized form in separate complexes with benzoate and acetate inhibitors and for the two-electron reduced form. Nitroreductase is a member of a group of enzymes that reduce a broad range of nitroaromatic compounds and has potential uses in chemotherapy and bioremediation. The monomers of the nitroreductase dimer adopt an α+β fold and together bind two flavin mononucleotide prosthetic groups at the dimer interface. In the oxidized enzyme, the flavin ring system adopts a strongly bent (16°) conformation, and the bend increases (25°) in the reduced form of the enzyme, roughly the conformation predicted for reduced flavin free in solution. Because free oxidized flavin is planar, the induced bend in the oxidized enzyme may favor reduction, and it may also account for the characteristic inability of the enzyme to stabilize the one electron-reduced semiquinone flavin, which is also planar. Both inhibitors bind over the pyrimidine and central rings of the flavin in partially overlapping sites. Comparison of the two inhibitor complexes shows that a portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for accommodating varied substrates. The crystal structure of the nitroreductase enzyme from Enterobacter cloacae has been determined for the oxidized form in separate complexes with benzoate and acetate inhibitors and for the two-electron reduced form. Nitroreductase is a member of a group of enzymes that reduce a broad range of nitroaromatic compounds and has potential uses in chemotherapy and bioremediation. The monomers of the nitroreductase dimer adopt an α+β fold and together bind two flavin mononucleotide prosthetic groups at the dimer interface. In the oxidized enzyme, the flavin ring system adopts a strongly bent (16°) conformation, and the bend increases (25°) in the reduced form of the enzyme, roughly the conformation predicted for reduced flavin free in solution. Because free oxidized flavin is planar, the induced bend in the oxidized enzyme may favor reduction, and it may also account for the characteristic inability of the enzyme to stabilize the one electron-reduced semiquinone flavin, which is also planar. Both inhibitors bind over the pyrimidine and central rings of the flavin in partially overlapping sites. Comparison of the two inhibitor complexes shows that a portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for accommodating varied substrates. Nitroaromatic compounds are pervasive pollutants whose toxicity is generally the result of their enzymatic reduction to more reactive species (1.Spain J.C. Annu. Rev. Microbiol. 1995; 49: 523-555Crossref PubMed Scopus (613) Google Scholar, 2.Esteve-Nunez A. Caballero A. Ramos J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 335-352Crossref PubMed Scopus (368) Google Scholar, 3.Rodgers J.D. Bunce N.J. Water Res. 2001; 35: 2101-2111Crossref PubMed Scopus (270) Google Scholar). There is considerable interest in the flavin-containing nitroreductases that catalyze the reductive activation of nitrated aromatics, because of their central role in mediating nitroaromatic toxicity (4.McCalla D.R. Reuvers A. Kaiser C. Biochem. Pharmacol. 1971; 20: 3532-3537Crossref PubMed Scopus (44) Google Scholar, 5.Tokiwa H. Ohnishi Y. Crit. Rev. Toxicol. 1986; 17: 23-60Crossref PubMed Scopus (475) Google Scholar, 6.Rickert D.E. Drug. Metab. 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Microbiol. 1993; 59: 2520-2525Crossref PubMed Google Scholar), and their utility in activating prodrugs in directed anticancer therapies (11.Knox R.J. Friedlos F. Borland M.P. Cancer Metastasis Rev. 1993; 12: 195-212Crossref PubMed Scopus (120) Google Scholar, 12.Niculescu-Duvaz I. Friedlos F. Niculescu-Duvaz D. Davies L. Springer C.J. Anticancer Drug Des. 1999; 14: 517-538PubMed Google Scholar). The nitroreductase from Enterobacter cloacae(NR) 1The abbreviations used are: NRnitroreductase from E. cloacaeNfsAmajor oxygen-insensitive NADPH-dependent nitroreductase of E. coliNTRminor oxygen-insensitive NAD(P)H-dependent nitroreductase of E. coliFRase Imajor NAD(P)H:FMN oxidoreductase of V. fischeriNOXNADH oxidase of T. thermophilusFRPNADPH-dependent flavin reductase of V. harveyiEox/hqflavin two-electron reduction midpoint potential relative to the normal hydrogen electrodeEox/sqmidpoint potential for the oxidized and one-electron reduced pair, Esq/hq, midpoint potential for the one- and two-electron reduced pairPEGpolyethylene glycolr.m.s.d.root mean square deviation 1The abbreviations used are: NRnitroreductase from E. cloacaeNfsAmajor oxygen-insensitive NADPH-dependent nitroreductase of E. coliNTRminor oxygen-insensitive NAD(P)H-dependent nitroreductase of E. coliFRase Imajor NAD(P)H:FMN oxidoreductase of V. fischeriNOXNADH oxidase of T. thermophilusFRPNADPH-dependent flavin reductase of V. harveyiEox/hqflavin two-electron reduction midpoint potential relative to the normal hydrogen electrodeEox/sqmidpoint potential for the oxidized and one-electron reduced pair, Esq/hq, midpoint potential for the one- and two-electron reduced pairPEGpolyethylene glycolr.m.s.d.root mean square deviation catalyzes two-electron reduction of a variety of nitrated aromatics as well as quinones and flavins (13.Bryant C. DeLuca M. J. Biol. Chem. 1991; 266: 4119-4125Abstract Full Text PDF PubMed Google Scholar, 14.Bryant C. Hubbard L. McElroy W.D. J. Biol. Chem. 1991; 266: 4126-4130Abstract Full Text PDF PubMed Google Scholar, 15.Koder R.L. Miller A.-F. Biochim. Biophys. Acta. 1998; 1387: 395-405Crossref PubMed Scopus (104) Google Scholar, 16.Rafii F. Hansen E.B. Antimicrob. Agents Chemother. 1998; 42: 1121-1126Crossref PubMed Google Scholar). Indeed, this enzyme was first isolated from bacteria growing in a weapons storage dump, and can reduce trinitrotoluene (80.Bryant C.P. A Biochemical and Biophysical Investigation of Enterobacter cloacae NitroreductasePh.D. thesis. University of California at San Diego, San Diego, CA1990Google Scholar). NR reduces nitrobenzene to the corresponding hydroxylamine and derives reducing equivalents from reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), or other nicotinamides (15.Koder R.L. Miller A.-F. Biochim. Biophys. Acta. 1998; 1387: 395-405Crossref PubMed Scopus (104) Google Scholar) by means of a flavin mononucleotide cofactor (FMN) (13.Bryant C. DeLuca M. J. Biol. Chem. 1991; 266: 4119-4125Abstract Full Text PDF PubMed Google Scholar, 14.Bryant C. Hubbard L. McElroy W.D. J. Biol. Chem. 1991; 266: 4126-4130Abstract Full Text PDF PubMed Google Scholar). nitroreductase from E. cloacae major oxygen-insensitive NADPH-dependent nitroreductase of E. coli minor oxygen-insensitive NAD(P)H-dependent nitroreductase of E. coli major NAD(P)H:FMN oxidoreductase of V. fischeri NADH oxidase of T. thermophilus NADPH-dependent flavin reductase of V. harveyi flavin two-electron reduction midpoint potential relative to the normal hydrogen electrode midpoint potential for the oxidized and one-electron reduced pair, Esq/hq, midpoint potential for the one- and two-electron reduced pair polyethylene glycol root mean square deviation nitroreductase from E. cloacae major oxygen-insensitive NADPH-dependent nitroreductase of E. coli minor oxygen-insensitive NAD(P)H-dependent nitroreductase of E. coli major NAD(P)H:FMN oxidoreductase of V. fischeri NADH oxidase of T. thermophilus NADPH-dependent flavin reductase of V. harveyi flavin two-electron reduction midpoint potential relative to the normal hydrogen electrode midpoint potential for the oxidized and one-electron reduced pair, Esq/hq, midpoint potential for the one- and two-electron reduced pair polyethylene glycol root mean square deviation NR follows ping-pong bi-bi kinetics, and its FMN groups cycle between the oxidized neutral and reduced anionic states with an Eox/hq of −190 mV, near that of free FMN (81.Koder R.L. Purification, Cloning, and Characterization of an Oxygen-insensitive NAD(P)H Nitroreductase from Enterobacter cloacae Strain 96-3Ph.D. thesis. The Johns Hopkins University, Baltimore, MD1999Google Scholar). Single-electron redox chemistry and associated formation of the semiquinone are not stabilized. This results in NR's activity being “oxygen-insensitive,” in that the enzyme does not readily transfer one electron to molecular oxygen to form the superoxide radical (13.Bryant C. DeLuca M. J. Biol. Chem. 1991; 266: 4119-4125Abstract Full Text PDF PubMed Google Scholar,15.Koder R.L. Miller A.-F. Biochim. Biophys. Acta. 1998; 1387: 395-405Crossref PubMed Scopus (104) Google Scholar). Several other FMN-containing oxidoreductases have been identified that have similar broad substrate specificity ranges and also do not stabilize the semiquinone state of the bound flavin. They are homodimers that share a similar fold and key amino acids, although the amino acid identities conserved over the whole group are few (17.Zenno S. Saigo K. Kanoh H. Inouye S. J. Bacteriol. 1994; 176: 3536-3543Crossref PubMed Google Scholar). The minor O2-insensitive nitroreductase of Escherichia coli (NTR or NfsB), shares 88% sequence identity with NR (18.Zenno S. Koike M. Tanokura M. Saigo K. J. Biochem. (Tokyo). 1996; 120: 736-744Crossref PubMed Scopus (127) Google Scholar). The major NAD(P)H:FMN oxidoreductase of Vibrio fischeri(FRase I) shares only 33% sequence identity (17.Zenno S. Saigo K. Kanoh H. Inouye S. J. Bacteriol. 1994; 176: 3536-3543Crossref PubMed Google Scholar, 19.Inouye S. DEBS Lett. 1994; 347: 163-168Crossref PubMed Scopus (63) Google Scholar) whereas NADH oxidase from Thermus thermophilus (NOX) is even more remotely related (20.Park H.J. Reiser C.O. Kondruweit H. Erdmann R.D. Schmid M. Eur. J. Biochem. 1992; 205: 881-885Crossref PubMed Scopus (111) Google Scholar, 21.Park H.J. Kreutzer R. Reiser C.O. Sprinzl M. Eur. J. Biochem. 1993; 211: 909PubMed Google Scholar). Additional homologues include the major O2-insensitive nitroreductase of Escherichia coli (NfsA) (22.Zenno S. Koike H. Kumar A.N. Jayaraman R. Tanokura M. Saigo K. J. Bacteriol. 1996; 178: 4508-4514Crossref PubMed Scopus (156) Google Scholar) and NADPH:flavin oxidoreductase of Vibrio harveyi (FRP) (23.Lei B. Liu M. Huang S. Tu S.-C. J. Bacteriol. 1994; 176: 3552-3558Crossref PubMed Google Scholar). Crystal structures have been deposited and/or published for several members of this family in the oxidized state, and all show an FMN bound at the interface between monomers in each of two symmetry-related but independent active sites (24.Hecht H.J. Erdmann H. Park H.-J. Sprinzl M. Schmid R.D. Nat. Struct. Biol. 1995; 2: 1109-1114Crossref PubMed Scopus (93) Google Scholar, 25.Tanner J.J. Lei B. Tu S.-C. Krause K.L. Biochemistry. 1996; 35: 13531-13539Crossref PubMed Scopus (92) Google Scholar, 26.Koike H. Sasaki H. Kobori T. Zenno S. Saigo K. Murphy M.E. Adman E.T. Tanokura M. J. Mol. Biol. 1998; 280: 259-273Crossref PubMed Scopus (77) Google Scholar, 27.Parkinson G.P. Skelly J.V. Neidle S. J. Med. Chem. 2000; 43: 3624-3631Crossref PubMed Scopus (104) Google Scholar, 28.Kobori T. Sasaki H. Lee W.C. Zenno S. Saigo K. Murphy M.E. Tanokura M. J. Biol. Chem. 2001; 276: 2816-2823Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 29.Lovering A.L. Hyde E.I. Searle P.F. White S.A. J. Mol. Biol. 2001; 309: 203-213Crossref PubMed Scopus (91) Google Scholar). 2PDB entry 1NEC; H. J. Hecht, C. Bryant, H. Erdmann, H. Pelletier, and R. Sawaya, unpublished data. 2PDB entry 1NEC; H. J. Hecht, C. Bryant, H. Erdmann, H. Pelletier, and R. Sawaya, unpublished data. We have determined crystal structures of oxidized NR in complex with two different inhibitors of the first half reaction as well as the crystal structure of the reduced enzyme. 3Atomic coordinates and structure factors have been deposited with the RCSB Protein Data Bank accession numbers: 1KQC(NR-acetate complex), 1KQB (NR-benzoate complex), 1KQD (reduced NR). 3Atomic coordinates and structure factors have been deposited with the RCSB Protein Data Bank accession numbers: 1KQC(NR-acetate complex), 1KQB (NR-benzoate complex), 1KQD (reduced NR). This work represents the most extensive comparison of states yet published for an NR homologue and includes the first crystal structure of one of these enzymes in the reduced state. Details of the flavin conformation and inhibitor binding interactions provide novel insights into the bases for the oxygen insensitivity and substrate specificity range of this important family of enzymes. E. cloacaenitroreductase was overexpressed in Escherichia coli and purified according to published methods (30.Koder R.L. Miller A.-F. Protein Expr. Purif. 1998; 13: 53-60Crossref PubMed Scopus (18) Google Scholar). The purified enzyme was stored at a concentration of 4.75 mg/ml in 50 mmKH2PO4 (pH 7), 0.02% (w/v) NaN3. The protein buffer was exchanged with 10 mm HEPES (pH 7) and 50 mm KCl prior to crystallization. Crystals of oxidized NR were grown by hanging-drop vapor diffusion at 4 °C against well solution containing 100 mm homopipes (pH 4.8), 25 mm acetate, and 15% PEG-4000. They grew to full size (0.5 × 0.5 × 0.1 mm) within 30 days. Prior to data collection, crystals were dialyzed against cryoprotectant solution containing 100 mm HEPES (pH 4.8), 25 mm sodium acetate, 25% PEG-4000, and 18% glycerol for 16 h. The crystals were mounted in nylon loops and flash cooled by plunging into liquid nitrogen (31.Rodgers D.W. Methods Enzymol. 1997; 276: 183-203Crossref PubMed Scopus (108) Google Scholar). Oxidized NR crystals were dialyzed against cryoprotectant solution containing 100 mmHEPES (pH 4.8), 600 mm sodium benzoate, 25% PEG-4000, and 18% glycerol for 20 h prior to flash cooling. A fresh solution of sodium dithionite was added to cryoprotectant, and 25 μl of the resulting solution was gently mixed with oxidized NR crystals in 25 μl of cryoprotectant. After ∼10 min, 25 μl of cryoprotectant buffer was removed and replaced with 25 μl of cryoprotectant buffer containing dithionite. This procedure was repeated two more times. The crystals lost their deep yellow color over the course of the treatment. They were then immediately flash cooled and stored in liquid nitrogen prior to data collection. Crystals were held at 115 K for data collection on a CuKα source with an R-AXIS IV++ image plate detector. Data were reduced with the HKL (32.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38440) Google Scholar) and CCP4 packages (33.CCP4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19729) Google Scholar). The crystal and data parameters are given in Table I. In all crystals there are four monomers, or two dimers, in the asymmetric unit.Table ISummary of crystallographic dataNR-oxidized acetateNR-oxidized benzoateNR reducedSpace groupP21P21P21Unit cella = 52.79, b = 79.61, c = 97.14a = 52.92, b = 79.39, c = 97.18a = 52.83, b = 79.98, c = 97.25β = 93.63β = 93.66β = 93.62Wavelength (Å)1.54181.54181.5418Resolution (Å)20.0–1.820.0–1.820.0–1.9Last shell (Å)1.88–1.801.88–1.801.99–1.90Average redundancy (last shell)4.77 (4.28)3.74 (3.51)4.00 (3.09)Rsym (last shell) (%)aRsym = ΣΣj‖Ij − 〈I〉‖/ΣIj.5.2 (27.8)6.5 (16.2)5.4 (14.3)I/ςI (last shell)28.3 (4.8)20.3 (8.3)27.6 (7.5)Completeness (last shell) (%)92.0 (87.5)97.5 (93.5)97.7 (88.9)a Rsym = ΣΣj‖Ij − 〈I〉‖/ΣIj. Open table in a new tab Initial structures were determined by molecular replacement using the CNS software package (34.Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J.N. Pannu N.S. Reed R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16947) Google Scholar) and the coordinate set of unliganded nitroreductase2 as a search object. Geometry restraint files for acetate and benzoate were taken from the HIC-UP site (35.Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1119-1131Crossref PubMed Scopus (496) Google Scholar). Initial restraint files for FMN were also taken from the HIC-UP site and modified to reflect theoretical values for oxidized and reduced flavin (36.Dixon D.A. Lindner D.L. Branchaud B. Lipscomb W.N. Biochemistry. 1979; 18: 5770-5775Crossref PubMed Scopus (59) Google Scholar). Individual weighting values for the FMN geometry parameters were adjusted to give stable refinement while still allowing the flavin to adopt the correct conformation (as judged by omit density and tests with low geometry weights and noncrystallographic symmetry restraints on the FMN groups to stabilize refinement). Initial manual rebuilding was followed by simulated annealing refinement in CNS and subsequent cycles of manual rebuilding (37.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar), addition of ordered solvent, and energy minimization. The resolution and quality of the data were sufficient for refinement without applying noncrystallographic symmetry restraints. Final refinement parameters are given in Table II.Table IISummary of refinementParameterNR-oxidized acetateNR-oxidized benzoateNR reducedResolution (Å)20.0–1.820.0–1.820.0–1.9Rwork/Rfree(%)aRwork, free = Σ∥Fobs‖ − ‖Fcalc∥/‖Fobs‖, for the reflections used in refinement (work) and the 10% of reflections held aside (free).18.8/21.918.9/21.818.8/22.0r.m.s.d. bond lengths (Å)0.0050.0050.005r.m.s.d. bond angles (Å)1.11.11.1r.m.s.d. improper angles (°)0.90.90.9r.m.s.d. dihedral angles (°)20.720.520.6B r.m.s.d. bonded atoms (main/side)1.1/1.91.1/1.81.1/1.9Average B(main/side) (Å2)15.7/16.814.9/16.115.3/16.5Average B all FMN (Å2)12.211.312.1Average B acetate or benzoate (Å2)30.914.4Number of solvent molecules449439438a Rwork, free = Σ∥Fobs‖ − ‖Fcalc∥/‖Fobs‖, for the reflections used in refinement (work) and the 10% of reflections held aside (free). Open table in a new tab NR (13.Bryant C. DeLuca M. J. Biol. Chem. 1991; 266: 4119-4125Abstract Full Text PDF PubMed Google Scholar, 14.Bryant C. Hubbard L. McElroy W.D. J. Biol. Chem. 1991; 266: 4126-4130Abstract Full Text PDF PubMed Google Scholar) and homologous enzymes (17.Zenno S. Saigo K. Kanoh H. Inouye S. J. Bacteriol. 1994; 176: 3536-3543Crossref PubMed Google Scholar, 18.Zenno S. Koike M. Tanokura M. Saigo K. J. Biochem. (Tokyo). 1996; 120: 736-744Crossref PubMed Scopus (127) Google Scholar, 21.Park H.J. Kreutzer R. Reiser C.O. Sprinzl M. Eur. J. Biochem. 1993; 211: 909PubMed Google Scholar, 38.Michael N.P. Brehm J.K. Anlezark G.M. Minton N.P. FEMS Microbiol. Lett. 1994; 124: 195-202Crossref PubMed Scopus (57) Google Scholar, 39.Watanabe M. Ishidate Jr., M. Nohmi T. Nucleic Acids Res. 1990; 18: 1059Crossref PubMed Scopus (47) Google Scholar) are dimers of 24-kDa subunits that share a characteristic α+β fold (Fig. 1). A central sheet consists of four antiparallel strands, with a fifth, parallel strand arising from the C terminus of the other dimer subunit. Surrounding the sheet are two large helices on one side, three smaller helices on the other, and two helices that pack against one end of the sheet. Several small helices or helical turns are also present. The FMN prosthetic groups bind in deep pockets at the dimer interface and interact with elements from both monomers (see Fig. 2 for FMN structure and numbering). In NR, each flavin group packs up against one end (S3) of the central sheet and is surrounded by helices H4 and H7. Helix H6 and the loop between H2 and S1 (particularly residues 36–43) from the other subunit form a cap over the cofactor binding site. H7 from that monomer also contributes to the pocket.Figure 2Chemical structure and atom numbering of the flavin mononucleotide prosthetic group of nitroreductase. a, oxidized flavin; b, fully reduced anionic form of the flavin. In both panels, the flavin is oriented as if viewing the re face. The other orientation is referred to as the si face.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Affinity for the FMN cofactor is high (10 nm), and within this structural family, binding typically involves extensive interactions with the protein, some of which are conserved or similar across the group. The polar groups in the isoalloxazine ring of FMN typically participate in a number of hydrogen bond interactions with protein, and seven hydrogen bonds with the flavin are present in the NR crystal structure. One group of contacts involves the N1, O4, and N3 positions of the ring system, including donation of a hydrogen bond by the backbone amide of Glu-165 to N5, which is thought to be the site of hydride transfer (40.Fraaije M.W. Mattevi A. Trends Biochem. Sci. 2000; 25: 126-132Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). This set of interactions is remarkably well conserved across the entire group of homologues, although the identities of some residues vary. One interacting residue, Gly-166 in NR, is absolutely conserved within the group. Main-chain torsion angles for this residue are either near the β-strand region of the Ramachandran plot (NR, NTR, FRase I, and NOX) or in the helical region (FRP and NfsA), both allowed positions for residues with side chains. Glycine is not, therefore, required at this position because of its greater backbone flexibility. Instead it may be conserved, because any side-chain atoms would intrude on space over the flavin reserved for substrate binding. The other group of contacts with the isoalloxazine ring involves the O2 and in some cases N1 atoms, which largely interact with basic residues. In NR and NTR, for example, there are two strong interactions between O2 of the pyrimidine ring and lysines 14 and 74. Unlike the first set of contacts, however, these interactions vary substantially across the group of related enzymes. This variation within the second group of interactions is particularly interesting, because the anionic forms of both the one- and two-electron reduced flavins develop resonance stabilized negative charge at the N1–C2=O2 locus. Positive electrostatic potential from the protein near this region is a common feature of flavoenzymes, and the degree of positive charge is thought to be a strong factor in determining the redox potential of the system (40.Fraaije M.W. Mattevi A. Trends Biochem. Sci. 2000; 25: 126-132Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 41.Ghisla S. Massey V. Eur. J. Biochem. 1989; 181: 1-17Crossref PubMed Scopus (492) Google Scholar). Variations in the exact nature of the contacts within the related group may then modulate the redox characteristics of the enzymes. In each of the NR homologues, the carbonyl oxygen from a residue located on the si side of the flavin interacts with the π system at the central ring (Fig. 3). The carbonyl oxygen is contributed by a proline residue (Pro-163) in NR. It is positioned about 2.8 Å from the plane of the central ring and almost directly below its center point, where the protein backbone runs across the short axis of the flavin. The rigid proline residue in NR is not required to place the carbonyl group in the observed orientation. Although both NTR and NOX also have prolines at this position, FRase I has a threonine (Thr-163), and NfsA and FRP both have tyrosines. As noted by others (42.Breinlinger E.C. Keenan C.J. Rotello V.M. J. Am. Chem. Soc. 1998; 120: 8606-8609Crossref Scopus (78) Google Scholar), this interaction of an electronegative group with the central region of the isoalloxazine ring is found in a number of flavin-containing proteins. The extensive interactions with the protein have a substantial effect on the conformation of the flavin. Both structural (43.Truss B.L. Fritchie C.J.J. Acta Crystallogr. Sect. B Struct. Sci. 1969; 25: 1911-1918Crossref PubMed Scopus (14) Google Scholar, 44.Norrestram R. Stensland B. Acta Crystallogr. Sect. B Struct. Sci. 1972; 28: 440-447Crossref Google Scholar, 45.Kuo M.C. Dunn B.R. Fritchie C.J.J. Acta Crystallogr. Sect. B Struct. Sci. 1974; 30: 1766-1771Crossref Google Scholar, 46.Porter D.J.T. Voet D. Acta Crystallogr. Sect. B Struct. Sci. 1978; 34: 598-610Crossref Google Scholar, 47.Fritchie C.J.J. Johnston R.M. Acta Crystallogr. Sect. B Struct. Sci. 1975; 31: 454-461Crossref Google Scholar, 48.Moonen C.T.W. Vervoort J. Müller F. Biochemistry. 1984; 23: 4859-4867Crossref PubMed Scopus (103) Google Scholar, 49.Wouters J. Moureau F. Perpète P. Norberg B. Evard G. Durant F. J. Chem. 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This distortion of the isoalloxazine has been termed butterfly bending to indicate a rigid motion of the two end rings about the N5–N10 axis. Both oxidized structures have the same overall bend of ∼16°, with an r.m.s. difference in atomic positions of only 0.03 Å. Although there have been other flavoproteins reported with similar or even more extreme butterfly bends, they are rare, probably comprising 10% or less of the reported structures (54.Lennon B.W. Williams C.H. Ludwig M.L. Protein Sci. 1999; 8: 2366-2379Crossref PubMed Scopus (108) Google Scholar). Crystallographic thermal factors indicate low mobility of the flavin, with an average on all atoms of 12.5 Å2 for the oxidized acetate complex and 11.4 Å2 for the oxidized benzoate complex. The flavin is rigidly fixed by interactions with the protein and is not disturbed by binding different ligands over the re face. It is likely that the geometry of the binding site plays a role in inducing the bent flavin conformation. The dimethylbenzene ring and a portion of the central ring are held tightly by structural elements on both faces. Hydrogen bonding groups interacting with the pyrimidine end of the isoalloxazine arise largely from the si side of the flavin, the same direction as the bend. On binding, then, the interactions with the pyrimidine ring are optimized at the cost of distorting the flavin. Bending of the flavin cofactor has also been attributed to steric aspects of the binding site in pyruvate oxidase (55.Müller Y.A. Schumacher G. Rudolph R. Schulz G.E. J. Mol. Biol. 1994; 237: 315-335Crossref PubMed Scopus (89) Google Scholar), NADH oxidase (24.Hecht H.J. Erdmann H. Park H.-J. Sprinzl M. Schmid R.D. Nat. Struct. Biol. 1995; 2: 1109-1114Crossref PubMed Scopus (93) Google Scholar), and in the reduced form of thioredoxin reductase (54.Lennon B.W. Williams C.H. Ludwig M.L. Protein Sci. 1999; 8: 2366-2379Crossref PubMed Scopus (108) Google Scholar). In addition, specific features of the protein-flavin interaction in NR may affect flavin conformation. Among the NR homologues, the interactions that stand out are: 1) the presence of an electron rich group (main-chain carbonyl oxygen in NR and its homologues) interacting with the π system of the isoalloxazine at or near the central ring, 2) a hydrogen bond donor (main-chain amide in NR and homologues) interacting with N5 of the flavin, 3) the presence of positively charged residues near the N1–C2=O2 locus, and 4) the absence of parallel π-π stacking of aromatic side chains with the flavin. To assess the significance of these interactions, we examined 54 flavoproteins in a unique data set identified by Lennon et al. (54.Lennon B.W. Williams C.H. Ludwig M.L. Protein Sci. 1999; 8: 2366-2379Crossref PubMed Scopus (108) Google Scholar) for the presence of each of the four interactions and grouped the data into three bend angle classes: 0–5°, 5–10°, and >10°. Only parallel stacking of the flavin with an aromatic group from the protein was clearly correlated with bend angle, being absent in all twelve structures in the highest bend angle group. All of the surveyed aromatic interactions involve stacking, at least in part, with the central ring of the isoalloxazine. Because this ring becomes significantly nonplanar and the π system weakens in a highly bent flav" @default.
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• W2061319036 date "2002-03-01" @default.
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• W2061319036 title "Structures of Nitroreductase in Three States" @default.
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• W2061319036 doi "https://doi.org/10.1074/jbc.m111334200" @default.
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