Crystal structure of reduced thioredoxin reductase from Escherichia coli: structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor.
ABSTRACT: Catalysis by thioredoxin reductase (TrxR) from Escherichia coli requires alternation between two domain arrangements. One of these conformations has been observed by X-ray crystallography (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816). This form of TrxR, denoted FO, permits the reaction of enzyme-bound reduced FAD with a redox-active disulfide on TrxR. As part of an investigation of conformational changes and intermediates in catalysis by TrxR, an X-ray structure of the FO form of TrxR with both the FAD and active site disulfide reduced has been determined. Reduction after crystallization resulted in significant local conformation changes. The isoalloxazine ring of the FAD cofactor, which is essentially planar in the oxidized enzyme, assumes a 34 degree "butterfly" bend about the N(5)-N(10) axis in reduced TrxR. Theoretical calculations reported by others predict ring bending of 15-28 degrees for reduced isoalloxazines protonated at N(1). The large bending in reduced TrxR is attributed in part to steric interactions between the isoalloxazine ring and the sulfur of Cys138, formed by reduction of the active site disulfide, and is accompanied by changes in the positions and interactions of several of the ribityl side-chain atoms of FAD. The bending angle in reduced TrxR is larger than that for any flavoprotein in the Protein Data Bank. Distributions of bending angles in published oxidized and reduced flavoenzyme structures are different from those found in studies of free flavins, indicating that the protein environment has a significant effect on bending.
Project description:Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 A; R = 22.2%; R(free) = 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 A; R = 24.1%; R(free) = 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.
Project description:The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD generating superoxide and a flavin radical that oxidize the isoalloxazine ring C7? methyl group and a nearby tyrosine residue. This tyrosine and key residues surrounding the oxygen pocket are conserved in enzymes from related bacteria, including pathogens such as Staphylococcus aureus. Photo-sensitivity may thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria.
Project description:Thioredoxin reductases (TrxRs) are flavin-containing dithioloxidoreductases that couple reduction equivalents from the soluble NAD(P)H pool to the soluble protein thioredoxin (Trx). Previous crystallographic studies of the Escherichia coli enzyme ( ecTrxR) have shown that low molecular weight TrxRs can adopt two distinct conformations: the first (FO) is required for the oxidation of the flavin cofactor and the generation of reduced Trx; the second (FR) is adopted for the reduction of the flavin by NAD(P)H. Here, protein electrochemistry has been used to interrogate the equilibrium between the oxidized and reduced conformations of the ecTrxR and a novel, low molecular weight TrxR from the thermophilic archaeon Thermoplasma acidophilum ( taTrxR) that is characterized structurally and biochemically in the accompanying paper [Hernandez et al. (2008) Biochemistry 47, 9728-9737]. A reversible electrochemical response is observed that reveals a dynamic behavior dependent upon the temperature of the experiment. At low temperatures (283 K) a broad, quasi-reversible electrochemical envelope is observed centered at a value of approximately -300 mV and displaying a peak width of over 150 mV. The voltammetric response sharpens dramatically as the temperature increases, becoming much more reversible (as determined by peak separation and peak width). The overall potential and shape of the voltammetric data indicate that the flavin (FAD/FADH 2) and disulfide/dithiol couples are very close in thermodynamic potentials, and the data are interpreted in terms of the model of two-state conformational change between flavin reducing (FR) and flavin oxidizing (FO) states, where the difference in potential for the flavin and disulfide cofactors must be within 40 mV of one another. In this model, the low temperature peak broadening is interpreted as an indication of a heterogeneous population of TrxR conformations that exist at low temperature; at higher temperatures, FO and FR conformers can rapidly interconvert, and voltammetry reports upon an average potential of the conformations.
Project description:Low G+C Gram-positive Firmicutes, such as the clinically important pathogens <i>Staphylococcus aureus</i> and <i>Bacillus cereus</i>, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from <i>S. aureus</i> and <i>B. cereus</i>. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.
Project description:Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the alpha subunit and the third domain is made up entirely by the beta subunit. The N-terminal portion of the alpha subunit and the majority of the beta subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the alpha subunit. Alignment of all the known sequences for the ETF alpha subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of alpha T266, suggesting why the pathogenic mutation, alpha T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4'-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between alpha H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.
Project description:Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.
Project description:Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme. Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx. The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP(+) has been determined to 3.0-A resolution by x-ray crystallography. The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR. The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. The C-terminal extension typical of mammalian TrxRs has two functions: (i) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and (ii) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide. Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart. This evolutionary switch renders cell growth dependent on selenium.
Project description:Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the d-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH- co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25 A. The structures of Klebsiella pneumoniae mutase with FAD and with FADH- bound have been determined to 2.2 A and 2.35 A resolution, respectively. This is the first report of the FADH(-)-containing structure. Two flavin-dependent mechanisms for the enzyme have been proposed, one, which involves a covalent adduct being formed at the flavin and the other based on electron transfer. Using our structural data, we have examined the two mechanisms. The electron transfer mechanism is consistent with the structural data, not surprisingly, since it makes fewer demands on the precise positioning of atoms. A model based on a covalent adduct FAD requires repositioning of the enzyme active site and would appear to require the isoalloxazine ring of FADH- to buckle in a particular way. However, the FADH- structure reveals that the isoalloxazine ring buckles in the opposite sense, this apparently requires the covalent adduct to trigger profound conformational changes in the protein or to buckle the FADH- opposite to that seen in the apo structure.
Project description:Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of proline to ?(1)-pyrroline-5-carboxylate, which is the first step of proline catabolism. Here, we report the structures of proline dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the proline analogue L-tetrahydrofuroic acid and in the reduced state with the proline site vacant. The analogue binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by helix ?8 and the ?1-?1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of ?8 by 1.7 Å, reconfiguration of the ?1-?1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases the catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases the efficiency 140-fold, which suggests that flexibility of the ?1-?1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition.
Project description:Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s(-1). However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s(-1) at air saturation) and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While ?-mercaptoethanol is a very poor substrate of sfALR (?0.3 min(-1) at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be physiologically important in preventing the oxidase from catalyzing the potentially harmful oxidation of intracellular glutathione.