Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: evidence that cysteine-138 functions to initiate dithiol-disulfide interchange and to accept the reducing equivalent from reduced flavin.

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys138 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.     

Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.     

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.     

Crystal structure of reduced thioredoxin reductase from Escherichia coli: structural flexibility in the isoalloxazine ring of the flavin adenine dinucleotide cofactor

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.     

Thioredoxin and peptide methionine sulfoxide reductase: convergence of similar structure and function in distinct structural folds.

Thioredoxin (Trx) and peptide methionine sulfoxide reductase (PMSR) are small thiol oxidoreductases implicated in antioxidant defense and redox regulation of cellular processes. Here we show that the structures of Trx and PMSR exhibit resemblance in their alphabeta core regions and that the active site cysteines in two proteins occupy equivalent positions downstream of a central beta-strand and at the N-terminus of an alpha-helix. Moreover, we identified a PMSR subfamily that contains an active site CxxC motif (two cysteines separated by two other amino acids) positioned similarly to the catalytic redox active CxxC motif in Trx. However, Trx and PMSR are characterized by distinct ancient folds that differ in both orientation of secondary structures and their patterns. Trx is a member of the Trx-fold superfamily, whereas PMSR has a unique fold not found in other proteins. The data suggest that similar structures and functions of Trx and PMSR were acquired independently during evolution and point to a general strategy of identifying new redox regulatory proteins.     

E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide

The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low K(M) value of 10 microM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in k(cat)/K(MetSO). Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.     

Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

人硫氧还蛋白在大肠杆菌中的高效表达、纯化及对内毒素血症小鼠的保护作用

为了对人硫氧还蛋白hTRX功能及其在休克治疗前景进行评价 ,将PCR扩增的hTRX基因连入pKPL 4载体 ,转化大肠杆菌pop2 136 .通过两步离子交换柱分离得到纯化的蛋白 ,进一步观察其稳定性及在体外对NFS 6 0和MCF 7细胞的促增殖活性 .结果发现 ,TRX蛋白可明显促进去细胞因子诱导的NFS 6 0细胞增殖和撤血清后MCF 7细胞的增殖 ,并发现外源性TRX可明显对小鼠内毒素血症起保护作用 .     

Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor.

Membrane-bound nitrate reductase of Escherichia coli consists of three subunits designated as A, B, and C, with subunit C being the apoprotein of cytochrome b, A hemA mutant that cannot synthesize delta-aminolevulinic acid (ALA) produces a normal, stable, membrane-bound enzyme when grown with ALA. When grown without ALA, this mutant makes a reduced amount of membrane-bound enzyme that is unstable and contains no C subunit. Under the same growth conditions, this mutant accumulates a large amount of a soluble form of the enzyme in the cytoplasm. Accumulation of this cytoplasmic form begins immediately upon induction of the enzyme with nitrate. The cytoplasmic form is very similar to the soluble form of the enzyme obtained by alkaline heat extraction. It is a high-molecular-weight complex with a Strokes radius of 8.0 nm and consists of intact A and B subunits. When ALA is added to a culture growing without ALA, the cytoplasmic form of the enzyme is incorporated into the membrane in a stable form, coincident with the formation of functional cytochrome b. Reconstitution experiments indicate that subunit C is present in cultures grown without ALA but is reduced in amount or unstable. These results indicate that membrane-bound nitrate reductase is synthesized via a soluble precursor containing subunits A and B, which then binds to the membrane upon interaction with the third subunit, cytochrome b.     

TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7

Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.     

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Partial 1H NMR assignments of the Escherichia coli dihydrofolate reductase complex with folate: evidence for a unique conformation of bound folate

Sequence-specific 1H assignments have been made for over 25% of the amino acid side chains of Escherichia coli dihydrofolate reductase complexed with folate by using a variety of two-dimensional techniques. Proton resonances were assigned by using a combination of site-directed mutagenesis and a knowledge of the X-ray crystal structure. Unique sets of NOE connectivities present in hydrophobic pockets were matched with the X-ray structure and used to assign many of the residues. Other residues, particularly those near or in the active site, were assigned by site-directed mutagenesis. The ability to assign unambiguously the proton resonances of these catalytically important residues allowed for extensive networks of NOE connectivities to follow from these assignments. As a consequence of these assignments, the orientation of the pterin ring of folate could be determined, and its conformation is similar to that of the productive dihydrofolate complex. Under these experimental conditions, only one bound form of the pterin ring could be detected.     

Redox interconversion of glutathione reductase from Escherichia coli. A study with pure enzyme and cell-free extracts

Summary The glutathione reductase from E. coli was rapidly inactivated following aerobic incubation of the pure and cell-free extract enzymes with NADPH, NADH and other reductants. The inactivation of the pure enzyme depended on the time and temperature of incubation (t_1/2 = 2 min at 37°C), and was proportional to the |INADPH|/|enzyme| ratio, reaching 50% in the presence of 0.3 M NADPH and 45 M NADH respectively, at a subunit concentration of 20 nM. Higher pyridine nucleotide concentrations were required to inactivate the enzyme from cell-free extracts. Two apparent pKa, corresponding to pH 5.8 and 7.3, were determined for the redox inactivation. The enzyme remained inactive even after eliminating the excess NADPH by gel chromatography. E. coli glutathione reductase was protected by oxidized and reduced glutathione against redox inactivation with both pure and cell-free extract enzymes. Ferricyanide and dithiothreitol protected only the pure enzyme, while NADP⁺ exclusively protected the cell-free extract enzyme. The inactive glutathione reductase was reactivated by treatment with oxidized and reduced glutathione, ferricyanide, and dithiothreitol in a time-and temperature-dependent process. The oxidized form of glutathione was more efficient and specific than the reduced form in the protection and reactivation of the pure enzyme.The molecular weight of the redox-inactivated E. coli glutathione reductase was similar to that of the dimeric native enzyme, ruling out aggregation as a possible cause of inactivation. A tentative model is discussed for the redox inactivation, involving the formation of an erroneous disulfide bridge at the glutathione-binding site.     

Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.