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.     

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.     

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.     

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.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Probing the Role of the Internal Disulfide Bond in Regulating Conformational Dynamics in Neuroglobin

In this report, we demonstrate that the internal disulfide bridge in human neuroglobin modulates structural changes associated with ligand photo-dissociation from the heme active site. This is evident from time-resolved photothermal studies of CO photo-dissociation, which reveal a 13.4± 0.9 mL mol⁻¹ volume expansion upon ligand photo-release from human neuroglobin, whereas the CO dissociation from rat neuroglobin leads to a significantly smaller volume change (ΔV = 4.6 ± 0.3 mL mol⁻¹). Reduction of the internal disulfide bond in human neuroglobin leads to conformational changes (reflected by ΔV) nearly identical to those observed for rat Ngb. Our data favor the hypothesis that the disulfide bond between Cys⁴⁶ and Cys⁵⁵ modulates the functioning of human neuroglobin.     

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.     

Structural and Kinetic Analysis of Free Methionine-R-sulfoxide Reductase from Staphylococcus aureus: CONFORMATIONAL CHANGES DURING CATALYSIS AND IMPLICATIONS FOR THE CATALYTIC AND INHIBITORY MECHANISMS*

Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys¹⁰² functions as the catalytic residue and Cys⁶⁸ as the resolving Cys that forms a disulfide bond with Cys¹⁰². Cys⁷⁸, previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97–106 containing the catalytic Cys¹⁰². We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.     

The mitochondrial glycine cleavage system

Summary The glycine cleavage enzyme system is composed of four different proteins tentatively called P-protein, H-protein, T-protein and L-protein, and catalyzes the following reaction reversibly: Glycine + tetrahydrofolate + NAD⁺ 5, 10-methylene-tetrahydrofolate + NH₃ + CO₂ + NADH + H^– Glycine decarboxylase, tentatively called P-protein, is able by itself to catalyze glycine decarboxylation, yielding methylamine as product, but at an extremely low rate. P-Protein alone is also able to catalyze slightly the exchange of carboxyl carbon of glycine with CO₂. However, the rates of the P-protein-catalyzed reactions are greatly increased by the co-existence of aminomethyl carrier protein, a lipoic acid-containing enzyme tentatively called H-protein. Several lines of evidence suggest that H-protein brings about a conformational change of P-protein which may be relevant to the expression of the decarboxylase activity of P-protein and that the functional glycine decarboxylase may be an enzyme complex composed of both P-protein and H-protein. H-Protein seems to play a dual role in the glycine decarboxylation; the one as a regulatory protein of P-protein, and the other as an electron-pulling agent and concomitantly as a carrier of the aminomethyl moiety derived from glycine. The idea that H-protein functions as a modulator of P-protein was further supported by the study of a patient with nonketotic hyperglycinemia. The primary lesion in this patient appeared to consist in structural abnormality in H-protein; the H-protein purified from the liver of this patient was apparently devoid of functional lipoic acid. Nevertheless, H-protein from the patient could stimulate the P-protein-catalyzed exchange of the carboxyl carbon of glycine and CO₂, although only to a limited extent. The observed activity should be independent of the functioning of lipoic acid and would be a reflection of a conformational change in P-protein brought about by H-protein.P-Protein was inactivated when it was incubated with glycine in the presence of II-protein, and the inactivation was completely prevented when bicarbonate was further added so as to allow the glycine-CO₂ exchange to proceed. The inactivation was accompanied by a spectral change of P-protein. The inactivation of P-protein seemed to take place as a side reaction of the glycine decarboxylation and to reflect the formation of a ternary complex of P-protein, H-protein and aminomethyl moiety of glycine through a Schiff base linkage of the H-protein-bound aminomethyl moiety with the pyridoxal phosphate of P-protein.     

Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14?Cys39 bond into the ovomucoid third domain from silver pheasant

P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴? Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴? Cys³⁹ bond into the flexible N‐terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH‐independent hydrolysis constant (K_hyd°) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K_hyd°, but has no effect on the thermal stability. The site‐specific disulfide introduction into the flexible N‐terminal loop of natural Kazal‐type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme

A three-disulfide form of hen egg white lysozyme with Cys⁶ and Cys¹²⁷ blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at itspH optimum,pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy atpH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys⁶ and Cys¹²⁷. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions atpH 2.0 but theT _m of 3SS-lysozyme, 18.9°C, was found to be 34° lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, atpH 2.0 and 35°C, if the conformational chemical potential for the folded forms ofboth 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys⁶-Cys¹²⁷ disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys⁶ and Cys¹²⁷ destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.Cornell Biotechnology Army Research Office Predoctoral Fellow, 1986–1989.     

Evolution of Substrate Specificity within a Diverse Family of β/α-Barrel-fold Basic Amino Acid Decarboxylases: X-RAY STRUCTURE DETERMINATION OF ENZYMES WITH SPECIFICITY FOR l-ARGININE AND CARBOXYNORSPERMIDINE*

Pyridoxal 5′-phosphate (PLP)-dependent basic amino acid decarboxylases from the β/α-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-Å resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the β/α-barrel domain of one subunit and the β-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the β-barrel domain we refer to as the “specificity helix.” Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu³¹⁴, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5′-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.     

Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6'' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.     

Characteristics of Dihydroxyphenylalanine/5-Hydroxytryptophan Decarboxylase Activity in Brain and Liver of Cat

Abstract: Dihydroxyphenylalanine/5-hydroxytryptophan (DOPA/5-HTP) decarboxylase activity varied widely in different parts of the CNS, being highest in the neostriatum and lowest in the frontal cortex. The addition of 2.5 μ m -pyridoxal 5'-phosphate (PLP), the coenzyme, increased enzyme activity in brainstem and liver, while higher concentrations led to a decrease in activity. In brainstem, the addition of 1000 μ m PLP shows activity similar to that obtained without exogenous PLP. The effects of different monoamine oxidase (MAO) inhibitors on decarboxylase activity were demonstrated. Iproniazid phosphate and harmaline significantly decreased the decarboxylation in liver and brainstem, while pargyline inhibited only liver decarboxylation. Some decarboxylase inhibitors such as RO4–4602 and α-methyl DOPA, as well as piribedil, a dopaminergic receptors agonist, were added in vitro to measure their action on decarboxylase with or without exogenous PLP or with double concentrations of substrate (5-HTP). Piribedil (5000 μ m ) affected the enzymic reaction and triggered a higher inhibition in liver. Inhibition in brainstem needed less RO4–4602 (50 μ m ) than in liver (300 μ m ). Addition of PLP did not reverse this inhibition, while doubling the concentration of 5-HTP nullified the inhibitory effect in liver only. Inhibition induced by α-methyl DOPA (5 μ m ) was easily reversed by doubling the concentration of substrate. However, the presence of exogenous PLP restored the enzymic activity in liver only. We conclude from this work thus that the enzyme can decarboxylate its substrate without exogenous PLP, that MAO inhibitors might inhibit decarboxylase activity, and that decarboxylase inhibitors react differently when brain and liver are used as enzymic source. PLP seems to act as a protective agent on the active site of the enzyme in the brainstem and preferentially with the substrate in the liver.     

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in?vitro drug screening

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val²¹⁶→Cys²¹⁶ mutation and fluorophore labelling for in vitro drug screening. The Val²¹⁶ residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys²¹⁶ residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor''s role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val²¹⁶→Cys²¹⁶ mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening.     

Human Eosinophil Major Basic Protein 2: Location of Disulfide Bonds and Free Sulfhydryl Groups

Eosinophil granule major basic protein 2 (MBP2 or major basic protein homolog) is a paralog of major basic protein (MBP1) and, similar to MBP1, is cytotoxic and cytostimulatory in vitro. MBP2, a small protein of 13,433 Da molecular weight, contains 10 cysteine residues. Mass spectrometry shows two cystine disulfide linkages (Cys²⁰–Cys¹¹⁵ and Cys⁹²–Cys¹⁰⁷) and 6 cysteine residues with free sulfhydryl groups (Cys², Cys²³, Cys⁴², Cys⁴³, Cys⁶⁸, and Cys⁹⁶). MBP2, similar to MBP1, has conserved motifs in common with C-type lectins. The disulfide bond locations are conserved among human MBP1, MBP2 and C-type lectins.     

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys⁵⁹/Cys⁶⁴), and a C-terminal cysteine/selenocysteine motif (Cys⁴⁹⁷/Sec⁴⁹⁸). 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 Cys⁵⁹/Cys⁶⁴ dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys⁵⁹/Cys⁶⁴ dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys⁴⁹⁷/Sec⁴⁹⁸ 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 H₂O₂-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.     

A PDI-catalyzed thiol–disulfide switch regulates the production of hydrogen peroxide by human Ero1

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O₂) and releases hydrogen peroxide (H₂O₂), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys²⁰⁸–Cys²⁴¹ disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H₂O₂ production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O₂ is reduced to H₂O₂. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys²⁰⁸/Cys²⁴¹-dependent mixed-disulfide complex with Ero1α. The H₂O₂-detoxifying glutathione peroxidase 8 also binds to the Cys²⁰⁸/Cys²⁴¹ loop region. Supported by O₂ diffusion simulations, these data describe the first enzymatically controlled O₂ access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H₂O₂ production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.     

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C–S bond cleavage,substrate scope,and an unexpected role for the tetramer

Bacterial two-component flavin-dependent monooxygenases cleave the stable C–S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS⁻) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C–S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS⁻ closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD’s functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.