Antimicrobial garlic-derived diallyl polysulfanes: Interactions with biological thiols in Bacillus subtilis

BackgroundDiallylpolysulfanes are the key constituents of garlic oils, known to exhibit broad spectrum anticancer and antimicrobial activity. Studies in vitro, and in mammalian cells, have shown they react, via thiol-polysulfane exchange, with their major low molecular weight thiol, glutathione. However, there are no detailed reports of diallylpolysulfane effects on other common thiol metabolites (cysteine and coenzyme A) or major thiol cofactors (e.g. bacillithiol) that many Gram positive bacteria produce instead of glutathione.MethodsDiallylpolysulfanes were individually purified then screened for antimicrobial activity against Bacillus subtilis. Their impact on thiol metabolites (bacillithiol, cysteine, coenzyme A, protein thiols allyl thiols//persulfides) in B. subtilis cultures were analysed, by HPLC.ResultsDiallylpolysulfane bioactivity increased with increasing chain length up to diallyltetrasulfane, but then plateaued. Within two minutes of treating B. subtilis with diallyltrisulfane or diallyltetrasulfane intracellular bacillithiol levels decreased by ~90%. Cysteine and CoA were also affected but to a lesser degree. This was accompanied by the accumulation of allyl thiol and allyl persulfide. A significant level of protein-S-allylation was also detected.ConclusionsIn addition to the major low molecular weight thiol, diallylpolysulfanes can also have an impact on other thiol metabolites and protein thiols.General significanceThis study shows the rapid parallel impact of polysulfanes on different biological thiols inside Bacillus subtilis alongside the concomitant generation of allyl thiols and persulfides.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Analysis of biological thiols: derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.     

Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design

Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co-assemble in different H and L ratios to form 24-mer shell-like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure–function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L-rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H-ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron-induced protein thermal destabilization effect (ΔT_m up to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo- and hetero-polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L-rich ferritins may have great potential for biotechnological applications.     

Iron mineralization and core dissociation in mammalian homopolymeric H-ferritin: Current understanding and future perspectives

BackgroundThe mechanism of iron oxidation and core formation in homopolymeric H-type ferritins has been extensively studied in-vitro, so has the reductive mobilization of iron from the inorganic iron(III) core. However, neither process is well-understood in-vivo despite recent scientific advances.Scope of reviewHere, we provide a summary of our current understanding of iron mineralization and iron core dissolution in homopolymeric H-type ferritins and highlight areas of interest and further studies that could answer some of the outstanding questions of iron metabolism.Major conclusionsThe overall iron oxidation mechanism in homopolymeric H-type ferritins from vertebrates (i.e. human H and frog M ferritins) is similar, despite nuances in the individual oxidation steps due to differences in the iron ligand environments inside the three fold channels, and at the dinuclear ferroxidase centers. Ferrous cations enter the protein shell through hydrophilic channels, followed by their rapid oxidization at di‑iron centers. Hydrogen peroxide produced during iron oxidation can react with additional iron(II) at ferroxidase centers, or at separate sites, or possibly on the surface of the mineral core. In-vitro ferritin iron mobilization can be achieved using a variety of reducing agents, but in-vivo iron retrieval may occur through a variety of processes, including proteolytic degradation, auxiliary iron mobilization mechanisms involving physiological reducing agents, and/or oxidoreductases.General significanceThis review provides important insights into the mechanisms of iron oxidation and mobilization in homopolymeric H-type ferritins, and different strategies in maintaining iron homeostasis.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

A method for site‐specific labeling of multiple protein thiols

We present a generic method for the site‐specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is removed, making available a reactive dithiol site for labeling with a second probe. For proof‐of‐principle, single and triple Cys mutants of the sulphate binding protein of an ABC transporter were constructed. The closely spaced thiols were engineered on the basis of the crystal structure of the protein and placed in different types of secondary structure elements and at different spacing. We show that phenyl arsenic oxide is a good protecting group for thiols spaced 6.3–7.3 Å. Proteins were labeled with two different fluorescent labels and the labeling ratios were determined with UV‐Vis spectroscopy and MALDI‐Tof mass spectrometry. The average labeling efficiency was ～80% for the single thiol and 65–90% for the dithiol site.     

Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite possible involvement of S-nitrosothiols

Glyceryl trinitrate specifically required cysteine, whereas NaNO₂ at concentrations less than 10 mM required one of several thiols or ascorbate, to activate soluble guanylate cyclase from bovine coronary artery. However, guanylate cyclase activation by nitroprusside or nitric oxide did not require the addition of thiols or ascorbate. Whereas various thiols enhanced activation by nitropruside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO₂^? or nitric oxide, was a potent activator of guanylate cyclase. Similarly, micromolar concentrations of the S-nitroso derivatives of penicillamine, GSH and dithiothreitol, prepared by reacting the thiol with nitric oxide, activated guanylate cyclase. Guanylate cyclase activation by S-nitrosothiols resembled that by nitric oxide and nitroprusside in that activation was inhibited by methemoglobin, ferricyanide and methylene blue. Similarly, guanylate cyclase activation by glyceryl trinitrate plus cysteine, and by NaNO₂ plus either a thiol or ascorbate, was inhibited by methemoglobin, ferricyanide and methylene blue. These data suggest that the activation of guanylate cyclase by each of the compounds tested may occur through a common mechanism, perhaps involving nitric oxide. Moreover, these findings suggest that S-nitrosothiols could act as intermediates in the activation of guanylate cyclase by glyceryl trinitrate, NaNO₂ and possibly     

The Sulfonylurea-Inhibited NADH Oxidase Activity of HeLa Cell Plasma Membranes has Properties of a Protein Disulfide–Thiol Oxidoreductase with Protein Disulfide–Thiol Interchange Activity

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC₅₀ of ca. 30 nM. LY181984, with an EC₅₀ of 30 nM, also inhibited a protein disulfide–thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide–thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide–thiol interchange coupled to cell enlargement.     

Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

Determination of endogenous thiols and thiol drugs in urine by HPLC with ultraviolet detection

Analysis of urine for endogenous thiols and thiol drugs content by HPLC with ultraviolet detection is addressed. Other methodologies for detection and determination of thiols in urine are only mentioned. Outline of metabolism, role of main biological thiols in physiological and pathological processes and their reference concentrations in urine are presented. In particular, urine sample preparation procedures, including reduction of thiol disulfides, chemical derivatization and reversed-phase HPLC separation steps are discussed. Some experimental details of analytical procedures for determination of endogenous thiols cysteine, cysteinylglycine, homocysteine, N-acetylcysteine, thioglycolic acid; and thiol drugs cysteamine, tiopronin, d-penicillamine, captopril, mesna, methimazole, propylthiouracil and thioguanine are reviewed.     

The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.     

Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.     

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.     

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide sensor

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.     

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H₂O₂), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H₂O₂, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H₂O₂ production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.     

Plasma low-molecular-weight thiol/disulphide homeostasis as an early indicator of global and focal cerebral ischaemia

Objective: Recent studies have shown that cerebral ischaemia causes not only local, but also systemic oxidative stress. This leads to oxidation of thiol-containing compounds, including low-molecular-weight thiols (cysteine, glutathione, homocysteine and others). Therefore, the aim of this work was to verify the hypothesis that the thiol/disulphide homeostasis of low-molecular-weight thiols is disturbed in the early stages of cerebral ischaemia.

Methods: Two experimental rat models of ischaemia were used: a global model of vascular ischaemia (clamping the common carotid arteries?+?haemorrhage) and focal ischaemia (middle cerebral artery occlusion). The total levels of thiols and their reduced forms were measured before surgery and after 40 minutes of reperfusion (global) or 3?hours (focal) ischaemia.

Results: The global ischaemia model caused a marked (2.5–4 times, P?P?Discussion: These results suggest that plasma low-molecular-weight thiols are actively involved in oxidation reactions at early stages of cerebral ischaemia; therefore, their reduced forms or redox state may serve as a sensitive indicator of acute cerebrovascular insufficiency.     

Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa.     

Purification and properties of the mercuric-ion-binding protein MerP.

The gene merP, coding for a mercuric-ion-binding periplasmic protein (P protein), was cloned into the expression vector pCA. In an Escherichia coli strain bearing the resulting plasmid, the P protein constitutes about 20% of total soluble protein. P protein was purified using ammonium sulfate precipitation and two chromatography steps. Typical yields were 20-30 mg from 7.5 l bacterial culture. The protein is a monomer with a molecular mass of 7500 Da. The periplasmic signal peptide was processed identically in both the recombinant and the wild-type proteins. CD spectra of both proteins were identical and indicated that the structure is highly ordered, containing approximately 80% alpha-helix. Purification in the presence of excess cysteine resulted in a form of the protein containing two reduced thiols, in agreement with the published sequence which has two cysteine residues. When cysteine was omitted from the purification buffers, no reduced thiol groups could be detected suggesting that the cysteine residues are oxidized. Both of these forms of the protein were found to bind approximately five Hg2+ ions/protein molecule in an apparently non-specific manner. However, in the presence of external thiol compounds, the protein with reduced thiols bound only one Hg2+ ion/protein molecule with an apparent Kd of 3.7 +/- 1.3 microM. Under these conditions, the protein with oxidized thiols did not bind Hg2+. The possible physiological role of this protein in Hg2+ detoxification is discussed.