Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK_a value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK_a value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes.     

Characterization of 2-bromoethanesulfonate as a selective inhibitor of the coenzyme m-dependent pathway and enzymes of bacterial aliphatic epoxide metabolism

Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO(2) fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO(2)-fixing enzyme 2-KPCC.     

Characterization of five catalytic activities associated with the NADPH:2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase of the Xanthobacter strain Py2 epoxide carboxylase system

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by carboxylation to acetoacetate. Epoxypropane carboxylation is a minimetabolic pathway that requires four enzymes, NADPH, NAD(+), and coenzyme M (CoM; 2-mercaptoethanesulfonate) and occurs with the overall reaction stoichiometry: epoxypropane + CO(2) + NADPH + NAD(+) + CoM --> acetoacetate + H(+) + NADP(+) + NADH + CoM. The terminal enzyme of the pathway is NADPH:2-ketopropyl-CoM [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase (2-KPCC), an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase family of enzymes and that catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-CoM to form acetoacetate and CoM according to the reaction: 2-ketopropyl-CoM + NADPH + CO(2) --> acetoacetate + NADP(+) + CoM. In the present work, 2-KPCC has been characterized with respect to the above reaction and four newly discovered partial reactions of relevance to the catalytic mechanism, and each of which requires the formation of a stabilized enolacetone intermediate. These four reactions are (1) NADPH-dependent cleavage and protonation of 2-ketopropyl-CoM to form NADP(+), CoM, and acetone, a reaction analogous to the physiological reaction but in which H(+) is the electrophile; (2) NADP(+)-dependent synthesis of 2-ketopropyl-CoM from CoM and acetoacetate, the reverse of the physiologically important forward reaction; (3) acetoacetate decarboxylation to form acetone and CO(2); and (4) acetoacetate/(14)CO(2) exchange to form (14)C(1)-acetoacetate and CO(2). Acetoacetate decarboxylation and (14)CO(2) exchange occurred independent of NADP(H) and CoM, demonstrating that these substrates are not central to the mechanism of enolate generation and stabilization. 2-KPCC did not uncouple NADPH oxidation or NADP(+) reduction from the reactions involving cleavage or formation of 2-ketopropyl-CoM. N-Ethylmaleimide inactivated the reactions forming/using 2-ketopropyl-CoM but did not inactivate acetoacetate decarboxylation or (14)CO(2) exchange reactions. The biochemical characterization of 2-KPCC and the associated five catalytic activities has allowed the formulation of an unprecedented mechanism of substrate activation and carboxylation that involves NADPH oxidation, a redox active disulfide, thiol-mediated reductive cleavage of a C-S thioether bond, the formation of a CoM:cysteine mixed disulfide, and enolacetone stabilization.     

Redox Regulation of Transglutaminase 2 Activity

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys²³⁰, Cys³⁷⁰, and Cys³⁷¹ that is involved in oxidative inactivation of TG2. Within this triad, Cys³⁷⁰ was found to participate in disulfide bonds with both Cys²³⁰ and its neighbor, Cys³⁷¹. Notably, Ca²⁺ was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys²³⁰ appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys³⁷⁰–Cys³⁷¹ through formation of the Cys²³⁰–Cys³⁷⁰ disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys²³⁰ is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.     

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).     

A catalytic dyad modulates conformational change in the CO2-fixing flavoenzyme 2-ketopropyl coenzyme M oxidoreductase/carboxylase

2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO₂ and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H⁺ and CO₂ to favor the metabolically preferred carboxylation product, acetoacetate.     

The unique Phe–His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S–C bond cleavage

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S–S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His–Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe–His. Here, we propose that this difference is important for coupling carboxylation with C–S bond cleavage. We substituted the Phe–His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.     

Probing the disulfide folding pathway of insulin‐like growth factor‐I

The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin‐like growth factor (IGF)‐I which when refolded in vitro produces the native three‐disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF‐I which contains a 13‐amino acid N‐terminal extension and a charge mutation at position 3 (Long‐ [Arg³]IGF‐I). Unlike IGF‐I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long‐[Arg³]GF‐I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long‐[Arg³]IGF‐I and IGF‐I, we acid‐trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non‐native intermediates, three native‐like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long‐[Arg³]IGF‐I; a single‐disulfide Cys¹⁸–Cys⁶¹ intermediate, an intermediate with Cys¹⁸–Cys⁶¹ and Cys⁶–Cys⁴⁸ disulfide bonds and another with Cys¹⁸–Cys⁶¹ and Cys⁴⁷–Cys⁵² disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys¹⁸‐Cys⁶¹, Cys⁶‐Cys⁴⁸ intermediate forms the native structure, not by the direct formation of the last (Cys⁴⁷‐Cys⁵²) disulfide bond, but by rearrangement via the Cys¹⁸–Cys⁶¹ intermediate and a productive Cys¹⁸–Cys⁶¹, Cys⁴⁷–Cys⁵² intermediate. In this pathway, the last disulfide bond to form involves Cys⁶ and Cys⁴⁸. Finally, we apply this pathway to IGF‐I and conclude that the divergence in the in vitro folding pathway of IGF‐I is caused by non‐native interactions involving Glu³ that stabilize the alternative structure. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 693–703, 1999.     

Characterization of a Reduced Form of Plasma Plasminogen as the Precursor for Angiostatin Formation

Plasma plasminogen is the precursor of the tumor angiogenesis inhibitor, angiostatin. Generation of angiostatin in blood involves activation of plasminogen to the serine protease plasmin and facilitated cleavage of two disulfide bonds and up to three peptide bonds in the kringle 5 domain of the protein. The mechanism of reduction of the two allosteric disulfides has been explored in this study. Using thiol-alkylating agents, mass spectrometry, and an assay for angiostatin formation, we show that the Cys⁴⁶²-Cys⁵⁴¹ disulfide bond is already cleaved in a fraction of plasma plasminogen and that this reduced plasminogen is the precursor for angiostatin formation. From the crystal structure of plasminogen, we propose that plasmin ligands such as phosphoglycerate kinase induce a conformational change in reduced kringle 5 that leads to attack by the Cys⁵⁴¹ thiolate anion on the Cys⁵³⁶ sulfur atom of the Cys⁵¹²-Cys⁵³⁶ disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys⁵¹²-Cys⁵³⁶ allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys⁴⁶²-Cys⁵⁴¹ and Cys⁵¹²-Cys⁵³⁶ disulfides have −/+RHHook and −LHHook configurations, respectively, which are two of the 20 different measures of the geometry of a disulfide bond. Analysis of the structures of the known allosteric disulfide bonds identified six other bonds that have these configurations, and they share some functional similarities with the plasminogen disulfides. This suggests that the −/+RHHook and −LHHook disulfides, along with the −RHStaple bond, are potential allosteric configurations.     

Biochemical evidence that regulation of Ero1β activity in human cells does not involve the isoform-specific cysteine 262

In the ER (endoplasmic reticulum) of human cells, disulfide bonds are predominantly generated by the two isoforms of Ero1 (ER oxidoreductin-1): Ero1α and Ero1β. The activity of Ero1α is tightly regulated through the formation of intramolecular disulfide bonds to help ensure balanced ER redox conditions. Ero1β is less tightly regulated, but the molecular details underlying control of activity are not as well characterized as for Ero1α. Ero1β contains an additional cysteine residue (Cys²⁶²), which has been suggested to engage in an isoform-specific regulatory disulfide bond with Cys¹⁰⁰. However, we show that the two regulatory disulfide bonds in Ero1α are likely conserved in Ero1β (Cys⁹⁰–Cys¹³⁰ and Cys⁹⁵–Cys¹⁰⁰). Molecular modelling of the Ero1β structure predicted that the side chain of Cys²⁶² is completely buried. Indeed, we found this cysteine to be reduced and partially protected from alkylation in the ER of living cells. Furthermore, mutation of Cys¹⁰⁰–but not of Cys²⁶²–rendered Ero1β hyperactive in cells, as did mutation of Cys¹³⁰. Ero1β hyperactivity induced the UPR (unfolded protein response) and resulted in oxidative perturbation of the ER redox state. We propose that features other than a distinct pattern of regulatory disulfide bonds determine the loose redox regulation of Ero1β relative to Ero1α.     

Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.     

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys²²⁷ and Cys³⁶¹. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.     

Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-¹³C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys²⁸² and Cys²⁶⁵ are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys²⁶⁵ and Cys²⁸² varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys²⁶⁵ appears to be in a relatively hydrophilic, oxidizable environment compared with Cys¹²⁷ and Cys²⁸². Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.     

Mechanistic implications of the structure of the mixed-disulfide intermediate of the disulfide oxidoreductase, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

The structure of the mixed, enzyme-cofactor disulfide intermediate of ketopropyl-coenzyme M oxidoreductase/carboxylase has been determined by X-ray diffraction methods. Ketopropyl-coenzyme M oxidoreductase/carboxylase belongs to a family of pyridine nucleotide-containing flavin-dependent disulfide oxidoreductases, which couple the transfer of hydride derived from the NADPH to the reduction of protein cysteine disulfide. Ketopropyl-coenzyme M oxidoreductase/carboxylase, a unique member of this enzyme class, catalyzes thioether bond cleavage of the substrate, 2-ketopropyl-coenzyme M, and carboxylation of what is thought to be an enzyme-stabilized enolacetone intermediate. The mixed disulfide of 2-ketopropyl-coenzyme M oxidoreductase/carboxylase was captured through crystallization of the enzyme with the physiological products of the reaction, acetoacetate, coenzyme M, and NADP, and reduction of the crystals with dithiothreitol just prior to data collection. Density in the active-site environment consistent with acetone, the product of reductive decarboxylation of acetoacetate, was revealed in this structure in addition to a well-defined hydrophobic pocket or channel that could be involved in the access for carbon dioxide. The analysis of this structure and that of a coenzyme-M-bound form provides insights into the stabilization of intermediates, substrate carboxylation, and product release.     

Structural insight into activation mechanism of Toxoplasma gondii nucleoside triphosphate diphosphohydrolases by disulfide reduction

The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258–268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys²⁵⁸–Cys²⁶⁸ show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg²⁺ to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg⁴⁹² and Glu⁴⁹³ in NTPDase1 by glycines in NTPDase3.     

Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser⁶⁰ on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys¹²⁶–Cys¹⁹⁶), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys⁶⁰ and gp120 Cys¹²⁶, and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.     

The thioredoxin-mediated recycling of Arabidopsis thaliana GRXS16 relies on a conserved C-terminal cysteine

Background

Glutaredoxins (GRXs) are oxidoreductases involved in diverse cellular processes through their capacity to reduce glutathionylated proteins and/or to coordinate iron?sulfur (Fe-S) clusters. Among class II GRXs, the plant-specific GRXS16 is a bimodular protein formed by an N-terminal endonuclease domain fused to a GRX domain containing a ¹⁵⁸CGFS signature.

Redox Regulation of Methionine Aminopeptidase 2 Activity

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys²²⁸-Cys⁴⁴⁸ disulfide bond that has an −RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of −261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m⁻¹ s⁻¹. The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys²²⁸-Cys⁴⁴⁸ disulfide is at the rim of the active site and is only three residues distant from the catalytic His²³¹, which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency.     

The thiol:disulfide oxidoreductase DsbB mediates the oxidizing effects of the toxic metalloid tellurite (TeO32-) on the plasma membrane redox system of the facultative phototroph Rhodobacter capsulatus

The highly toxic oxyanion tellurite (TeO3(2-)) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbB(WT), in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an "electron conduit" between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of Te(IV), the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.     

Allosteric Control of βII-Tryptase by a Redox Active Disulfide Bond

The S1A serine proteases function in many key biological processes such as development, immunity, and blood coagulation. S1A proteases contain a highly conserved disulfide bond (Cys¹⁹¹–Cys²²⁰ in chymotrypsin numbering) that links two β-loop structures that define the rim of the active site pocket. Mast cell βII-tryptase is a S1A protease that is associated with pathological inflammation. In this study, we have found that the conserved disulfide bond (Cys²²⁰–Cys²⁴⁸ in βII-tryptase) exists in oxidized and reduced states in the enzyme stored and secreted by mast cells. The disulfide bond has a standard redox potential of −301 mV and is stoichiometrically reduced by the inflammatory mediator, thioredoxin, with a rate constant of 350 m⁻¹ s⁻¹. The oxidized and reduced enzymes have different substrate specificity and catalytic efficiency for hydrolysis of both small and macromolecular substrates. These observations indicate that βII-tryptase activity is post-translationally regulated by an allosteric disulfide bond. It is likely that other S1A serine proteases are similarly regulated.