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• W2003676718 abstract "Escherichia coli 2,4-dienoyl-CoA reductase is an iron-sulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another iron-sulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP+ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis- and 2-trans,4-trans-dienoyl-CoA thioesters. Escherichia coli 2,4-dienoyl-CoA reductase is an iron-sulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another iron-sulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP+ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis- and 2-trans,4-trans-dienoyl-CoA thioesters. Metabolism of unsaturated fatty acids requires auxiliary enzymes in addition to those used in β-oxidation. After a given number of cycles through the β-oxidation pathway, those unsaturated fatty acyl-CoAs with double bonds at even-numbered carbon positions contain 2-trans,4-cis double bonds that cannot be modified by enoyl-CoA hydratase. Therefore, an auxiliary enzyme, 2,4-dienoyl-CoA reductase (DCR 1The abbreviations used are: DCR, 2,4-dienoyl-CoA reductase; TMDh, trimethylamine dehydrogenase; SAD, single wavelength anomalous; OYE, old yellow enzyme.1The abbreviations used are: DCR, 2,4-dienoyl-CoA reductase; TMDh, trimethylamine dehydrogenase; SAD, single wavelength anomalous; OYE, old yellow enzyme.; EC 1.3.1.34), is used that utilizes NADPH to remove the C4-C5 double bond (1Dommes V. Kunau W.H. J. Biol. Chem. 1984; 259: 1781-1788Abstract Full Text PDF PubMed Google Scholar, 2You S.Y. Cosloy S. Schulz H. J. Biol. Chem. 1989; 264: 16489-16495Abstract Full Text PDF PubMed Google Scholar). DCR is unusual in that it lacks stereospecificity, catalyzing the reduction of both natural fatty acids with cis double bonds, as well as substrates containing trans double bonds (3Cuebas D. Schulz H. J. Biol. Chem. 1982; 257: 14140-14144Abstract Full Text PDF PubMed Google Scholar). Two structurally unrelated forms of DCR have been isolated, both of which use NADPH as reducing equivalents to catalyze reduction of a 2,4-dienoyl-CoA to an enoyl-CoA. The Escherichia coli enzyme contains both FMN and FAD noncovalently bound to a single polypeptide, reducing the substrate by a hydride transfer mechanism in which reducing equivalents from NADPH are supplied indirectly to substrate (1Dommes V. Kunau W.H. J. Biol. Chem. 1984; 259: 1781-1788Abstract Full Text PDF PubMed Google Scholar). It functions as a monomer, having a molecular mass of 73 kDa, and has been shown recently to contain a 4Fe-4S cluster (4Liang X. Thorpe C. Schulz H. Arch. Biochem. Biophys. 2000; 380: 373-379Crossref PubMed Scopus (21) Google Scholar). The enzyme produces 2-trans-enoyl-CoA (1Dommes V. Kunau W.H. J. Biol. Chem. 1984; 259: 1781-1788Abstract Full Text PDF PubMed Google Scholar), which can be incorporated into the β-oxidation pathway. In contrast, three homologous eukaryotic forms have been identified, two mitochondrial and one peroxisomal. Two of these lack flavin and iron-sulfur cofactors but are homotetramers with a total molecular mass of 124 kDa. (The third has not been characterized.) The substrate is reduced by a direct hydride transfer mechanism from NADPH to form trans-3-enoyl-CoA (1Dommes V. Kunau W.H. J. Biol. Chem. 1984; 259: 1781-1788Abstract Full Text PDF PubMed Google Scholar). Thus, eukaryotic DCR requires another auxiliary enzyme, Δ3,Δ2-enoyl-CoA isomerase, to convert trans-3-enoyl-CoA to trans-2-enoyl-CoA (1Dommes V. Kunau W.H. J. Biol. Chem. 1984; 259: 1781-1788Abstract Full Text PDF PubMed Google Scholar), which can be subsequently fed into the β-oxidation pathway. Lastly, eukaryotic DCR has been shown to metabolize fatty acids with double bonds at odd as well as even positions (5Smeland T.E. Nada M. Cuebas D. Schulz H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6673-6677Crossref PubMed Scopus (77) Google Scholar, 6He X.Y. Shoukry K. Chu C. Yang J. Sprecher H. Schulz H. Biochem. Biophys. Res. Commun. 1995; 215: 15-22Crossref PubMed Scopus (36) Google Scholar), whereas there is currently no evidence to support this for the E. coli enzyme. In addition to DCR from E. coli, sequence analysis shows both Gram-negative and Gram-positive bacteria to have open reading frames encoding for DCR-like proteins; for example Yersinia pestis and Streptomyces coelicolor proteins have 72 and 62% sequence identities, respectively, with E. coli DCR. These conserved residues include the four cysteine residues that coordinate the iron-sulfur cluster and the cofactor binding sequence motifs. E. coli DCR also shares homology with a large family of TIM barrel-containing flavoenzymes, including the single-domain TIM barrel enzyme NADH oxidase from Thermoanaerobium brockii, which shares a 30% identity and much larger multi-domained proteins such as trimethylamine dehydrogenase (TMDh) from Methylotrophus methylophilus; this enzyme also contains a 4Fe-4S cluster and shares a 26% sequence identity with DCR spanning the entire length of both polypeptides (7He X.Y. Yang S.Y. Schulz H. Eur. J. Biochem. 1997; 248: 516-520Crossref PubMed Scopus (28) Google Scholar). A preliminary report on the structure of a functional homolog of DCR, 2-enoate reductase from Clostridium tyrobutyricim, has been presented (8Steinbacher S. Stumpf M. Weinkauf S. Rohdich F. Bacher A. Simon H. Chapman S. Perham R. Scrutton N. Flavins and Flavoproteins 2002. Agency for Scientific Publications, Berlin2002: 941-949Google Scholar). This enzyme shares a 32% sequence identity with E. coli DCR, binding FMN, FAD, and a 4Fe-4S cluster; however, 2-enoate reductase differs from DCR because it functions as a dodecamer, uses NADH rather than NADPH as an electron source, and reduces 2-enoates that are not activated by conjugation to CoA. Because the 2-enoate reductase structure reported lacks a bound substrate analog, little is known about its enzyme mechanism, including how the flavin and iron-sulfur cofactors participate in electron transfer from NAD(P)H to substrate. To this end, the crystal structure of E. coli DCR has been solved in the presence of substrate and gives insights into the catalytic and electron transfer mechanisms of prokaryotic forms of DCR. Protein Purification—Protein was expressed and purified as described previously (7He X.Y. Yang S.Y. Schulz H. Eur. J. Biochem. 1997; 248: 516-520Crossref PubMed Scopus (28) Google Scholar). Briefly, plasmid pNDH, which encodes E. coli DCR, was transformed into E. coli BL21(DE3)pLysS. Cells were cultured in Luria broth medium and induced by isopropyl-1-thio-β-d-galactopyranoside before being pelleted and resuspended by sonication into a 20 mm phosphate buffer solution, pH 7.4, including 10 mm β-mercaptoethanol. The suspension was centrifuged, and the supernatant was loaded onto a DEAE-cellulose column (Sigma). A 100 mm phosphate buffer solution, pH 7.4, including 10 mm β-mercaptoethanol was used to elute protein, which was subsequently transferred to a 2′,5′-ADP-Sepharose 4B column (Amersham Biosciences). Protein was eluted with a 20 mm phosphate buffer solution containing 5 μm 2-trans,4-trans-decadienoyl-CoA and 10 mm β-mercaptoethanol; the free ligand was removed by passing through another DEAE-cellulose column. Protein was stored at –80 °C in 20% glycerol, 100 mm phosphate buffer, pH 7.4, 1 mm EDTA, and 5 mm β-mercaptoethanol. Protein Crystallization—Purified protein was crystallized by vapor diffusion using the hanging-drop method (9McPherson A. Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 176-196Google Scholar), with the reservoir solution containing 30% polyethylene glycol 5000 monomethyl ether, 0.2 m sodium acetate, 0.1 m ammonium sulfate, and 0.1 m 2-(N-morpholino)ethane sulfonic acid, pH 6.5. Precipitant solution containing 18% polyethylene glycol 5000 monomethyl ether, 180 mm sodium acetate, 90 mm ammonium sulfate, and 90 mm 2-(N-morpholino)ethane sulfonic acid, pH 6.5, was mixed with protein at 15 mg/ml in a 1:1.5 volume ratio. Protein in storage solution was thawed and concentrated in the presence of a 2-fold molar excess of NADP+ before crystallization set-up. Rod-shaped crystals having a yellow/brown color appeared within about 1 week, reaching a maximum size of 40 × 40 × 500 μm. No cryoprotectant was used before flash cooling the crystals in liquid nitrogen. Crystals typically diffracted to 2.2 Å. Two heavy atom derivatives of DCR were used for phasing; first, a mercury derivative was made by co-crystallizing protein in the presence of a precipitant solution containing 10% saturated mercuric acetate. An osmium derivative was obtained by soaking crystals in reservoir solution containing 100% saturated osmium hexachloride for 10 min. In both cases, no additional cryo-protectant was used prior to cryo-cooling. Data Collection and Structure Determination—A complete native data set of DCR was collected in-house using an R-AXIS IIC image plate equipped with a Rigaku RU 200 generator, an MSC X-stream cooling system set at –180 °C, and Osmic blue confocal mirrors. High resolution data were collected at the Advanced Photon Source (Argonne, IL), beam-line 14-BM-C. Data were processed using the HKL suite of programs (10Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38555) Google Scholar). Difference Patterson map analysis was performed in XtalView (11McRee D.E. Practical Protein Crystallography. Academic Press, San Diego1999Google Scholar), and phases were calculated using either SHARP (12La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar) (for single wavelength anomalous (SAD) phases) or SOLVE (13Terwilliger T.C. Berendzen J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar) (for multiple isomorphous replacement with anomalous scattering phases). Cross-Fourier calculations for locating additional heavy atom sites were done using PHASES (14Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar), and phase combination of SAD and multiple isomorphous replacement with anomalous scattering phases was carried out using CNS (15Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kuntsleve 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 Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16963) Google Scholar). The resulting phases were improved by density modification using SOLOMON (16Abrahams J.P. Leslie A.G.W. Acta Crystallogr. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar), as implemented in SHARP, with subsequent inclusion of partial structure. Model building was initially done using Xfit (11McRee D.E. Practical Protein Crystallography. Academic Press, San Diego1999Google Scholar), with the final stages in O (17Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). Multiple cycles of refinement were performed in CNS, with rigid body refinement, simulated annealing, and positional refinement used in the initial stages, and positional and B-factor refinement during later stages of refinement. Water molecules were included near the end of refinement, initially using the program DDQ (18van den Akker F. Hol W.G. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 206-218Crossref PubMed Scopus (48) Google Scholar), followed by manual addition in the O graphics program. Structure-based sequence alignment was performed using INDONESIA, 2Madsen, D., Johansson, P., and Kleywegt, G. J. Web address: xray.bmc.uu.se/~dennis. using the Levitt and Gerstein method (36Levitt M. Gerstein M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5913-5920Crossref PubMed Scopus (256) Google Scholar), followed by some manual adjustments. Electrostatic and surface calculations were performed using GRASP (19Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5315) Google Scholar). Electron transfer pathway calculations were made using HARLEM (20Curry W.B. Grabe M.D. Kurnikov I.V. Skourtis S.S. Beratan D.N. Regan J.J. Aquino A.J. Beroza P. Onuchic J.N. J. Bioenerg. Biomembr. 1995; 27: 285-293Crossref PubMed Scopus (73) Google Scholar). Structure Determination—Native crystals diffracted to 2.4 Å using the in-house x-ray source, with data collected at the synchrotron extending to 2.2 Å. The mercury derivative diffracted in-house to 2.7 Å, and the osmium derivative to 2.4 Å at the synchrotron. The Harker sections of the native anomalous difference Patterson map from in-house data showed a strong peak, above 10 σ, corresponding to the 4Fe-4S cluster; however, the resolution was insufficient to resolve individual iron peaks. SAD phasing was performed in SHARP using the four iron atoms from an ideal 4Fe-4S cluster, which were positioned to give the best visual agreement with the Patterson map. The phases were modified in SOLOMON, and the resulting map showed clear density indicating a large TIM barrel domain in the structure, with traceable density outlining two additional domains extending into other parts of the molecule. Cross-Fourier analysis of mercury and osmium derivatives showed them to contain one and two heavy atom sites per asymmetric unit, respectively. These data were scaled and phased in SOLVE using both isomorphous and anomalous components, then combined with the SAD phases from SHARP using the “combine” CNS script, prior to performing density modification in SOLOMON. These steps resulted in minor improvements in electron density for a medium-sized and a small domain, with the most pronounced improvement around what was subsequently shown to be the FAD moiety in the small domain. An initial Cα-atom trace was made, and this model was included in a further round of solvent flattening using SigmaA phase combination (21Read R.J. Acta Crystallogr. A. 1986; 42: 140-149Crossref Scopus (2035) Google Scholar). Subsequent poly-alanine and partial structure models were included in this phase refinement step to further improve the quality of the electron density map. Data collection and final refinement statistics are listed in Table I.Table IData collection and refinement statisticsNativeaData collected at Advanced Photon Source, beamline 14-BM-C.NativebData collected in-house.OsmiumaData collected at Advanced Photon Source, beamline 14-BM-C.MercurybData collected in-house.Data collectionWavelength (Å)0.901.540.901.54Unit cell dimensions (Å)a =65.7365.6065.4466.44b =108.47109.23108.39108.50c =110.51110.30109.67111.66Resolution (Å)2.2 (-2.26)2.4 (-2.48)2.4 (-2.47)2.7 (-2.75)Completeness (%)cValues in parentheses are from the highest resolution shell.94.8 (61.2)91.4 (71.4)83.7 (59.8)92.4 (68.1)Mean <I/σ(I)>cValues in parentheses are from the highest resolution shell.30.3 (7.8)43.8 (5.6)18.4 (4.2)10.3 (2.5)Total no. of reflections898148985006255483216402Unique no. of reflections42467318743192523012R sym (%)cValues in parentheses are from the highest resolution shell.5.7 (24.9)5.5 (18.4)7.0 (35.7)9.0 (25.3)R iso (%)11.028.3Phasing statisticsMethodSADMIRASdMIRAS, multiple isomorphous replacement with anomalous scattering.Resolution (Å)2.42.5eAutomatically set by SOLVE.No. of sites421Phasing power (cent./acent.)fcent./acent., centric/acentric.-/1.600.37/0.400.30/0.33FOMgFOM, figure of merit.0.28acent.0.22Combined FOMgFOM, figure of merit.0.36Refinement statisticsResolution (Å)2.2R cryst/R free (%)20.3/24.4Average B-factors (Å2)Protein (5097 atoms)38.4Solvent (352 atoms)41.0Others (206 atoms)48.1r.m.s. deviationBond lengths (Å)0.01Bond angles (°)1.4Ramachandran analysisMost favored (%)84.9Allowed (%)15.1a Data collected at Advanced Photon Source, beamline 14-BM-C.b Data collected in-house.c Values in parentheses are from the highest resolution shell.d MIRAS, multiple isomorphous replacement with anomalous scattering.e Automatically set by SOLVE.f cent./acent., centric/acentric.g FOM, figure of merit. Open table in a new tab Overall Protein Structure—The overall fold of the enzyme is composed of three domains: an N-terminal TIM barrel (residues 1–368) which binds FMN, the 4Fe-4S cluster, and the substrate; a flavodoxin-like fold (residues 369–467 and 626–671) which binds FAD; and an NADP(H)-binding domain (residues 468–625) (Fig. 1). These domains will be referred to as the N-terminal, middle, and C-terminal domains, respectively. There are two disordered regions including residues 370–372 and residues 452–455, both of which form part of the loops on the surface of the protein; however, both N and C termini are well defined, with the C terminus ending as an α-helix. The arrangement of the three domains and the overall polypeptide fold resemble that seen in TMDh (22Lim L.W. Shamala N. Mathews F.S. Steenkamp D.J. Hamlin R. Xuong N.H. J. Biol. Chem. 1986; 261: 15140-15146Abstract Full Text PDF PubMed Google Scholar). The structure-based sequence alignment with TMDh shows DCR to have an α-helical insert in the C-terminal domain, which forms part of the protein's surface (Fig. 2). A large loop, which forms part of the dimer interface of TMDh in the C-terminal domain (residues 607–630), is missing in DCR, as is the C-terminal extension. Superimposition of DCR with one monomer of TMDh gives an r.m.s. deviation of ∼1.5 Å over 467 Cα carbon atoms (corresponding mostly to residues within the N-terminal and middle domains), showing that the high degree of sequence similarity extends to structural similarity. The FMN and 4Fe-4S cofactors that appear in both proteins are spatially conserved, and the bound ADP of TMDh occupies the same spatial position as the ADP portion of FAD in DCR. The conserved cysteine residues (334, 337, 341, and 353) coordinate the four iron atoms of the iron-sulfur cluster (Fig. 2).Fig. 2Structure-based sequence alignment of DCR (dcr) with TMDh (tmdhase). Their corresponding secondary structure elements are included. The resulting sequence alignment was fixed, and the sequence of enoate reductase (eno_red) was included to improve accuracy of the alignment with DCR. Black and white boxes show identical and homologous residues, respectively. Gray boxes indicate cysteine residues that coordinate the iron-sulfur cluster; the two black chevrons highlight the catalytic residues of DCR. The figure was generated using ESPript (33Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2525) Google Scholar). Every 10 residues of the DCR sequence are marked, and residue numbers at the end of each line for all sequences are included.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Active Site—Inspection of an [|Fo | – |Fc |] map over the si-face of the isoalloxazine ring of FMN in the early stages of refinement showed long tubular electron density, indicating the presence of substrate/product bound to the active site. This density could be fitted with one molecule of substrate, 2-trans,4-trans-decadienoyl-CoA (used for elution from the affinity column during protein purification), which was included in the model for subsequent rounds of model building and refinement. However, what was surprising during the mid-stages of model building was that the density bifurcated in the middle of the acyl chain. Further rounds of model building and refinement showed this fork to stem from the C5 atom of the acyl chain of the substrate, and that the C4-C5 double bond had been reduced by β-mercaptoethanol (included during protein purification and also in the storage buffer). The strong electron density in the [|Fo | – |Fc |] map corresponding to the sulfur atom of β-mercaptoethanol allowed correct placement of the remaining acyl chain and of β-mercaptoethanol, showing the product to have R-configuration at the chiral center (carbon 5). Fig. 3A shows an OMIT map confirming bifurcation at the C5 atom. The carbon-carbon double bond of substrate that is reduced during catalysis (atoms C4-C5 of the acyl chain) is sandwiched between the si-face of the isoalloxazine ring of FMN, centered around atom N5, and two residues of the polypeptide chain, Tyr-166 and His-252. The phenolic oxygen atom of Tyr-166 is about 3.5 Å away from the C4 atom of substrate, whereas the Cϵ atom of the imidazole ring of His-252 is in van der Waals contact with the C2 and C3 atoms of substrate, and its Nϵ atom forms a hydrogen bond to Tyr-166. These residues form part of a binding pocket illustrated in Fig. 3B. Interactions between substrate and protein at the catalytic site are outlined in Figs. 4 and 5A.Fig. 5Balls-and-sticks representation of the binding sites of substrate (A), FMN (B), and FAD (C). For clarity, only those residues that form hydrogen bonds with the ligand/cofactor are shown; those residues that form significant van der Waals interactions are discussed in the text. For simplicity, the diagram shows a flattened projection with water molecules removed. The figure was generated using LIGPLOT (34Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4349) Google Scholar) and rendered using MOLSCRIPT (35Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). Light gray balls represent carbon atoms, mid-gray are oxygen, dark gray are nitrogen, large mid-dark gray balls are sulfur, and black balls are phosphorus.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Substrate Binding Site— Fig. 5A illustrates the substrate binding site. The catalytic residue His-252 forms a hydrogen bond with the pantothenate region of the substrate, with the Nδ atom of the imidazole ring being 3.1 Å from the N4 atom of the pantothenate region. Three additional hydrogen bonds are formed between the protein and the pantothenate moiety of the substrate; a carbonyl backbone contact from His-252 to N8, the indole ring of Trp-577 to O9, and the guanido group of Arg-255 to O10, respectively. The ADP moiety of substrate is bound to a relatively basic region on the protein's surface, with the 3′-phosphate in close proximity to Arg-580 and Lys-566; the latter residue forms a salt bridge. However, the high B-factors (∼95 Å2) of the phosphate atoms suggest that this is a relatively weak association. The adenine ring is sandwiched between Pro-122 and Arg-255 and forms a hydrogen bond from the N6 atom to the guanido group of Arg-175, whereas the pyrophosphate linkage is exposed to solvent. The α-adenylic phosphate forms an intramolecular hydrogen bond to the O10 atom of the pantothenate chain. Glu-164 forms a hydrogen bond to the thioester carbonyl oxygen atom of the acyl chain, which may promote the positive character of the C5 atom of the acyl chain through resonance of the conjugated double bonds. FMN Binding Site—As seen in Fig. 1, the FMN cofactor is well buried within the protein's core. The si-face of the isoalloxazine ring is centered against the C3 atom of the acyl chain of substrate, with atoms C2 and C4 in van der Waals contact distance with the N10 (3.6 Å) and N5 (3.4 Å) atoms of FMN, respectively (Fig. 4). Met-25 is positioned on the opposing re-face of the isoalloxazine ring. Fig. 5B shows residues that interact with the FMN cofactor. Unlike TMDh in which the isoalloxazine ring of FMN is tethered to the polypeptide through a covalent bond between Cys-30 and the C6 atom of FMN, the FMN of DCR is not covalently attached to the enzyme; His-26 replaces the thiol residue of TMDh (Fig. 2). The ribityl chain forms hydrogen bonds with the guanidinium group of Arg-214 and the backbone carbonyl oxygen atom of Ser-24. The guanidinium group of Arg-214 also hydrogen bonds with the O2 atom of the isoalloxazine ring, as does the amide side chain of Gln-100. The N3 atom of the isoalloxazine ring forms a hydrogen bond to an amide group of Gln-100, and the O4 atom hydrogen bonds to the backbone nitrogen atom of Gly-58. Oxidized FMN is able to hydrogen bond to the backbone nitrogen atom of His-26 from the N5 atom of the isoalloxazine ring. The phosphate group is surrounded by backbone nitrogen atoms of Arg-288, Ala-310, and Arg-311. The guanido group of Arg-311 also forms hydrogen bonds to the phosphate. FAD Binding Site—Similar to FMN, the isoalloxazine ring of FAD is well buried; however, the ribityl and pyrophosphate moieties project to the protein surface such that the adenosine region is exposed to solvent (Fig. 1). The isoalloazine ring is positioned such that the two phenyl groups of Phe-412 and Phe-424 and the methyl side chain of Ala-415 are on its si-face, with the phenyl groups pointing edge-on to the plane of the isoalloxazine ring, and the nicotinamide and adjacent ribose ring of NADP(H) are on its re-face. There are no hydrogen bonds between protein or water molecules and the ribityl chain of FAD (Fig. 5C); however, the O3′ atom of the ribityl chain forms a hydrogen bond with the β-adenylic phosphate group in the pyrophosphate linkage. Both phosphate groups of the pyrophosphate linkage make several hydrogen bonds with backbone nitrogen atoms, Ala-384, Gln-411, and Gly-648, with the γ-amide side chain of Gln-411 also forming a hydrogen bond to the α-adenylic phosphate. Both hydroxyl groups of the adenosyl ribose ring form hydrogen bonds with the carboxylate of Asp-403, whereas the N1 and N6 atoms of the adenine ring form hydrogen bonds with backbone atoms of Val-448. NADP(H) Binding Site—Superimposition of the DCR structure with that of glutathione reductase shows that the middle and C-terminal domains of DCR resemble the N-terminal and middle domains of glutathione reductase (23Karplus P.A. Schulz G.E. J. Mol. Biol. 1987; 195: 701-729Crossref PubMed Scopus (432) Google Scholar), having an r.m.s. deviation of 1.6 Å for ∼43% of Cα carbon atoms in the two domains of glutathione reductase. The superimposition shows both FAD and NADP(H) occupying spatially conserved sites. During the mid-stages of model building of DCR, there was sufficient electron density above the re-face of the FAD flavin moiety to allow NADP+ to be included in the model. Unfortunately, however, it was not possible to accurately position the nicotinamide ring because there was not enough electron density to unambiguously fit the ring. Subsequent rounds of refinement failed to improve density surrounding this part of the molecule, although the well-defined electron density of the adjacent ribose ring and the surrounding peptide limits positioning of the nicotinamide ring to a small area. Therefore, it remains unclear whether the A- or B-side of the nicotinamide ring interacts with the re-face of the FAD isoalloxazine ring. Electron Transfer Pathway— Fig. 4 shows the arrangement of cofactors and substrates found in the crystal structure. The relative positions of the electron source (NADPH) and the electron sink (substrate) suggest that electrons from NADPH must travel through FAD, 4Fe-4S, and FMN to reduce substrate. Inter-cofactor distances appear substantially short enough to allow rapid electron transfer, with the FAD to 4Fe-4S minimal distance being 8.6 Å between N3 of the isoalloxazine ring and the nearest iron atom, sufficiently below the 14 Å electron tunneling limit proposed by Page et al. (24Page C.C. Moser C.C. Chen X. Dutton P.L. Nature. 1999; 402: 47-52Crossref PubMed Scopus (1510) Google Scholar). The distance between the iron-su" @default.
• W2003676718 created "2016-06-24" @default.
• W2003676718 creator A5015376852 @default.
• W2003676718 creator A5037798237 @default.
• W2003676718 creator A5061137007 @default.
• W2003676718 creator A5061517807 @default.
• W2003676718 date "2003-09-01" @default.
• W2003676718 modified "2023-10-03" @default.
• W2003676718 title "The Crystal Structure and Reaction Mechanism of Escherichia coli 2,4-Dienoyl-CoA Reductase" @default.
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