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 Å; R ؍ 22.2%; Rfree ؍ 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 Å; 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.hioredoxins are the major cellular protein disulfide reductases and are responsible for the regulation of numerous biochemical processes within the cell (1). These proteins are maintained in a reduced state by thioredoxin reductases (TrxR), homodimeric f lavoproteins that catalyze the NADPHdependent reduction of thioredoxins (2, 3).Two forms of TrxRs have evolved with related but distinct modes of catalysis (2-5). Low-M r TrxRs (M r Ϸ 35 kDa) are typically found in prokaryotes, archaea, plants, and lower eukaryotes, whereas high-M r TrxRs (M r Ϸ 55 kDa) are observed in higher eukaryotes. To date, only the green algae Chlamydomonas reinhardtii has been shown to contain both a low-and a high-M r TrxR (6).The general features of catalysis are retained in both low-and high-M r TrxR (2, 4). TrxR transfers reducing equivalents from NADPH to its bound FAD, ultimately leading to the reduction of an active-site disulfide. In low-M r TrxRs, the catalytic cycle requires a large conformational change after dithiol activation (4,7,8). In high-M r TrxR, the active-site dithiol reduces a third redox active center in the highly mobile C terminus of the opposing subunit. This third group is responsible for the reduction of the disulfide bond within thioredoxin. Its nature is species-specific and ranges from a C-X-X-X-X-C disulfide in Plasmodium falciparum (9) to a vicinal disulfid...
Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity. In contrast to the previously reported structure of lipovitellin, which has a funnel-like lipid-binding cavity, the lipid-binding site is encompassed in a β-sandwich formed by 2 β-sheets from the C-terminal domain of MTPα. The lipid-binding cavity of MTPα is large enough to accommodate a single lipid. PDI independently has a major role in oxidative protein folding in the endoplasmic reticulum. Comparison of the mechanism of MTPα binding by PDI with previously published structures gives insights into large protein substrate binding by PDI and suggests that the previous structures of human PDI represent the “substrate-bound” and “free” states rather than differences arising from redox state.
A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.
Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The ratelimiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.Glutathione, ␥-glutamylcysteinyl glycine, is a low molecular weight thiol, central to maintenance of redox homeostasis. Among its normal functions are the scavenging of reactive oxygen and nitrogen species (1), storage and transport of cysteine (2, 3), leukotriene, and prostaglandin biosynthesis (4, 5), and regulation of enzyme activity via reduction of disulfide bonds and glutathionylation (6, 7). Disruption of glutathione metabolism is associated with the progression of AIDS, cancer, and neurodegenerative conditions such as Parkinson and Alzheimer disease (8 -12). Polymorphisms that reduce activity of glutamate cysteine ligase (GCL), 2 the first and rate-limiting enzyme in de novo synthesis of glutathione, are correlated with reduced glutathione levels in patients with hemolytic anemia, schizophrenia and other neurological disorders (13-16). Given its importance both in normal and disease states, there is considerable interest in the development of novel compounds that could be used to modulate intracellular glutathione levels, potentially via GCL.Glutathione is synthesized from its three constituent amino acids by consecutive action of two cytosolic ATP-dependent enzymes: GCL and glutathione synthetase (17). GCL catalyzes the conjugation of the ␥-carboxyl group of L-glutamate to the amino group of L-cysteine (17). The proposed catalytic mechanism proceeds via phosphorylation of the ␥-carboxylate of L-glutamate by ATP (18 -20). The ␣-amino group of L-cysteine acts as a nucleophile, attacking the ␥-glutamyl phosphate intermediate to produce ␥-glutamylcysteine. This dipeptide is then coupled in an analogous fashion to glycine by glutathione synthetase to generate glutathione. As the committed step of glutathione biosynthesis, GCL activity is regulated by L-cysteine availability (21), feedback inhibition by glutathione (22), and transcriptional and post-translational regulation (23).Based on sequence analysis, three distinct groups of GCL have been identified. Groups 1 and 3 are comprised of bacterial and plant orthologues (24). Recently, x-ray crystal structures of Escherichia coli (Group 1) (25) and Brassica juncea (Group 3) (26) GCL were described, providing the first structural insights into ␥-glutamylc...
Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.
Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 Å ; R ؍ 19.9%, R free ؍ 25.1%) indicates that the inhibitor occupies both the glutamate-and the presumed cysteine-binding site and disrupts the previously observed Mg 2؉ coordination in the ATP-binding site. L-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 Å ; R ؍ 18.1%, R free ؍ 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme. Glutamate cysteine ligase (GCL)2 catalyzes the initial and rate-limiting step of glutathione biosynthesis (1, 2). The ATPdependent mechanism proceeds via a ␥-glutamylphosphate intermediate (2-4), with a subsequent nucleophilic attack by the ␣-amino group of L-cysteine to produce ␥-glutamylcysteine (1, 2). There are three distinct families of GCL enzymes: ␥-proteobacteria (Group 1), nonplant eukaryotes (Group 2), and ␣-proteobacteria and plants (Group 3) (5). Despite low sequence conservation between these groups (typically Ͻ10% sequence identity), all of the GCL appear to use this general catalytic mechanism. The resulting ␥-glutamylcysteine is coupled to L-glycine by glutathione synthetase (1) in an analogous reaction to generate reduced GSH, an abundant cellular reducing agent.GCL activity is tightly modulated by free L-cysteine availability (6), transcriptional regulation (7), and post-translational modifications (8). In addition, GCL is feedback regulated by the end product, glutathione (9). Glutathione inhibits GCL competitively with respect to L-glutamate, suggesting that the two binding sites are coincident (9). In heterodimeric GCL, such as the Drosophila, rat, and human enzymes, binding of the modifier subunit relieves feedback inhibition both by increasing the K i for glutathione and decreasing the K m for glutamate (10 -13). Further studies with glutathione analogues such as ophthalmic acid, S-methylglutathione, and GSSG have demonstrated that the free thiol group of glutathione is necessary for maximal inhibition (1, 9). However, the precise mode of glutathione binding has not been described.The central role of GCL in glutathione homeosta...
Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 sec −1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 Å; R factor =17.9%; R free =20.8%) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole. Keywordsγ-glutamyltranspeptidase; Ntn-hydrolase; glutathione; acivicin; x-ray crystallography H. pylori γ-glutamyltranspeptidase (HpGT) is a γ-glutamyl hydrolase with broad substrate specificity (1,2), and is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily (3,4). The inactive precursor undergoes an intramolecular autoprocessing event, generating the mature and catalytically active heterotetramer. A conserved threonine residue, Thr 380, serves as the N-terminal nucleophile and is required for both maturation and enzymatic activity (1).HpGT has been shown to degrade extracellular glutathione and glutamine, providing a growth advantage to the bacterium within its microenvironment (2,5,6). Similarly, upregulation of human γ-glutamyltranspeptidase in cancer is thought to help supplement these rapidly dividing cells with essential amino acid precursors for glutathione and protein biosynthesis (7,8). In mammalian systems, γ-glutamyltranspeptidase has been shown to be critical for the transport of cysteine for use in protein and glutathione biosynthesis (9,10). The enzyme is required for normal glutathione metabolism, initiating extracellular glutathione degradation. Subsequent steps lead to the cellular uptake of the composite amino acids of glutathione: glutamate, cysteine, and glycine (11,12).Acivicin is a com...
NHLRC2 (NHL repeat-containing protein 2) is an essential protein. Mutations of NHLRC2, including Asp148Tyr, have been recently associated with a novel FINCA disease (fibrosis, neurodegeneration, cerebral angiomatosis), which is fatal in early childhood. To gain insight into the mechanisms of action of this essential protein, we determined the crystal structure of the Trx-like and NHL repeat β-propeller domains of human NHLRC2 to a resolution of 2.7 Å. The structure reveals two domains adjacent to each other that form a cleft containing a conserved CCINC motif. A SAXS structure of full-length NHLRC2 reveals that the non-conserved C-terminal domain does not pack against the N-terminal domains. Analysis of the surface properties of the protein identifies an extended negative electrostatic potential in the surface of the cleft formed by the two domains, which likely forms a binding site for a ligand or interaction partner(s). Bioinformatics analysis discovers homologs across a range of eukaryotic and prokaryotic species and conserved residues map mostly to the adjacent surfaces of the Trx-like and β-propeller domains that form the cleft, suggesting both that this forms the potential functional site of NHLRC2 and that the function is conserved across species. Asp148 is located in the Trx-like domain and is not conserved across species. The Asp148Tyr mutation destabilizes the structure of the protein by 2°C. The NHLRC2 structure, the first of any of its homologs, provides an important step towards more focused structure-function studies of this essential protein.
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