The innate immunity role of cathepsin-D is linked to Trp-491 and Trp-492 residues of listeriolysin O

Listeriolysin O (LLO) is a thiol-activated cytolysin secreted by Listeria monocytogenes . LLO and phosphatidylinositol phospholipase C are two essential virulence factors, which this bacterium needs to escape from the phagosomal compartment to the cytoplasm. Cathepsin-D specifically cleaves LLO, between the Trp-491 (tryptophan amino acid in three letter nomenclature) and Trp-492 residues of the conserved undecapeptide sequence, ECTGLAWEWWR, in the domain 4 of LLO (D4). Moreover, these residues also correspond to the phagosomal-binding epitope. Cathepsin-D had no effect on phosphatidylinositol phospholipase C. We have observed that cathepsin-D cleaved the related cholesterol-dependent cytolysin pneumolysin at the same undecapeptide sequence between Trp-435 and Trp-436 residues. These studies also revealed an additional cathepsin-D cleavage site in the pneumolysin D4 domain localized in the 361-GDLLLD-366 sequence. These differences might confer a pathogenic advantage to listeriolysin O, increasing its resistance to phagosomal cathepsin-D action by reducing the number of cleavages sites in the D4 domain. Using ΔLLO/W491A and ΔLLO/W492A bacterial mutants, we reveal that the Trp-491 residue has an important role linked to cathepsin-D in Listeria innate immunity.     

Listeriolysin O-dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines

The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system. Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis. By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we found that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotactic protein-1) and NF-kappa B nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothelial cell activation. Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)-deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes. Adhesion molecule expression, chemokine secretion and NF-kappa B activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO. The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene. NF-kappa B activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non-haemolytic isogenic mutant. Direct evidence that LLO is involved in NF-kappa B activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries. Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process.     

Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.     

Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis

Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.     

The RTX haemolysins ApxI and ApxII are major virulence factors of the swine pathogen Actinobacillus pleuropneumoniae: evidence from mutational analysis

The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin-deficient mutants constructed by a mini-Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini-Tn10 were cloned and sequenced. In the non-haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non-haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.     

Surface-modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry.

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine-free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys-58, Cys-85, Cys-136, Cys-148 and Cys-334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin-state equilibria, NADH turnover rates and camphor-hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox-active label, N-ferrocenylmaleimide, to the single surface cysteine mutant SCF-K344C, and the electrochemistry of this modified mutant are also described.     

A novel topology and redox regulation of the rat brain K+-dependent Na+/Ca2+ exchanger,NCKX2

In this study we have examined the roles of endogenous cysteine residues in the rat brain K(+)-dependent Na(+)/Ca(2+) exchanger protein, NCKX2, by site-directed mutagenesis. We found that mutation of Cys-614 or Cys-666 to Ala inhibited expression of the exchanger protein in HEK-293 cells, but not in an in vitro translation system. We speculated that Cys-614 and Cys-666 might form an extracellular disulfide bond that stabilized protein structure. Such an arrangement would place the C terminus of the exchanger outside the cell, contrary to the original topological model. This hypothesis was tested by adding a hemagglutinin A epitope to the C terminus of the protein. The hemagglutinin A epitope could be recognized with a specific antibody without permeabilization of the cell membrane, supporting an extracellular location for the C terminus. Additionally, the exchanger molecule could be labeled with biotin maleimide only following extracellular application of beta-mercaptoethanol. Surprisingly, mutation of Cys-395, located in the large intracellular loop, to Ala, prevented reduction-dependent labeling of the protein. The activity of wild-type exchanger, but not the Cys-395 --> Ala mutant, was stimulated after application of beta-mercaptoethanol. Co-immunoprecipitation experiments demonstrated self-association between wild-type and FLAG-tagged exchanger proteins that could not be inhibited by Cys-395 --> Ala mutation. These results suggest that NCKX2 associates as a dimer, an interaction that does not require, but may be stabilized by, a disulfide linkage through Cys-395. This linkage, perhaps by limiting protein mobility along the dimer interface, reduces the transport activity of NCKX2.     

Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes

A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes. The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes. Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence. Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial -virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. -monocytogenes.     

Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.

Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.     

Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin.

The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.     

Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase.

In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.     

Cloning of the phycobilisome rod linker genes from the cyanobacterium Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Coexpression of pairs of nonhaemolytic H1yA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one H1yA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a non-haemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.     

Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin

By using a photoactivatable analog of 11-cis-retinal in rhodopsin, we have previously identified the amino acids Phe-115, Ala-117, Glu-122, Trp-126, Ser-127, and Trp-265 as major sites of cross-linking to the chromophore. To further investigate the amino acids that interact with retinal, we have now used site-directed mutagenesis to replace a variety of amino acids in the membrane-embedded helices in bovine rhodopsin, including those that were indicated by cross-linking studies. The mutant rhodopsin genes were expressed in monkey kidney cells (COS-1) and purified. The mutant proteins were studied for their spectroscopic properties and their ability to activate transducin. Substitution of the two amino acids, Trp-265 and Glu-122 by Tyr, Phe, and Ala and by Gln, Asp and Ala, respectively, resulted in blue-shifted (20-30 nm) chromophore, and substitution of Trp-265 by Ala resulted in marked reduction in the extent of chromophore regeneration. Light-dependent bleaching behavior was significantly altered in Ala-117----Phe, Trp-265----Phe, Ala, and Ala-292----Asp mutants. Transducin activation was reduced in these mutants, in particular Trp-265 mutants, as well as in Glu-122----Gln, Trp-126----Leu (Ala), Pro-267----Ala (Asn, Ser), and Tyr-268----Phe mutants. These findings indicate that Trp-265 is located close to retinal and Glu-122, Trp-126, and probably Tyr-268 are also likely to be near retinal.     

Vacuolar H+-ATPase and weak base action in Dictyostelium

Infection of a murine-spleen dendritic cell line by Listeria monocytogenes was found to induce cell death through apoptosis. To characterize the bacterial product(s) involved in induction of apoptosis, dendritic cells were infected with the L. monocytogenes EGD strain and several isogenic mutants deficient in the production of individual listerial virulence factors. The ability to induce cellular apoptosis was retained by all mutants tested, except the prfA and Δhly mutants, both of which are unable to produce listeriolysin. Apoptosis was also induced by purified listeriolysin suggesting that this protein directly induces apoptosis. Purified recombinant listeriolysins rendered either weakly haemolytic by a C-484 to S mutation, or non-haemolytic by a W-491 to A mutation exhibited little or no capacity to induce apoptosis, indicating that both activities are associated within the same protein region. Treatment with purified listeriolysin or L. monocytogenes infection also triggers apoptosis in explanted bone-marrow dendritic cells. Thus invasion of dendritic cells by L. monocytogenes, which results in cell death, may play an important role in the pathogenesis of listerial infections by impairing immune responses, hindering bacterial clearance and promoting spread of the infection.     

Molecular characterisation of the haemolytic isolates of Bacillus anthracis originated in Poland

Bacillus anthracis is generally considered non-haemolytic, when cultured on the solid media. However, strains capable to lyse sheep erythrocytes have been reported. Anthrolysin O, an orthologue of cereolysin was proposed as a putative haemolysin of B. anthracis. AIM: to determine whether anthrolysin O, haemolytic enterotoxin HBL and the pleiotropic regulator PlcR that activates antrholysin O production are associated with a haemolytic activity of B. anthracis strains isolated in Poland. MATERIAL: in total 8 B. anthracis strains - the fully virulent BL1 and seven the pXO2 lacking strains including: a vaccine strain Sterne 34F2 together with three haemolytic and three non-haemolytic strains isolated from different samples of the same animal died from anthrax in Poland. METHODS: The haemolytic activity was detected using Columbia agar plates supplemented with 5% of sheep blood. Anthrolvsin O, cereolysin and gene hblA encoding the key subunit of the HBL were detected by PCR. In addition, the plcR gene fragment containing the B. anthracis specific non-sense mutation was analysed by the DNA sequencing. Ten marker loci based MLVA genotyping was performed to distinguish tested strains. RESULTS: The alo gene encoding anthrolysin O was detected in both the haemolytic and non-haemolytic strains while hblA was absent. The B. anthracis specific plcR non-sense mutation was detected in both the groups of tested strains, suggesting that the haemolysis in tested strains may rather be conferred by the PlcR-independent factors. Moreover, haemolytic and non-haemolytic strains were indistinguishable by the MLVA. Obtained results may argue the haemolytic and non-haemolytic strains are isogenic and most probably a single mutational event is responsible for the haemolytic phenotype induction.     

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis

Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.     

Amino acid residues critical for the specificity for betaine aldehyde of the plant ALDH10 isoenzyme involved in the synthesis of glycine betaine

Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K(m)(BAL) increases and V(max)/K(m)(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V(max)/K(m)(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.     

Isolation and selection of biosurfactant-producing bacteria

Haemolysis has been used as an initial selection criterion for the primary isolation of surfactant-producing bacteria. Only 37 of 492 strains of different origins had haemolytic activity. These 37 strains, together with 49 non-haemolytic ones chosen at random, were studied for surface activity. Only five strains, all of them haemolytic, tested positive. Haemolysis and biosurfactant-production are thus probably associated.     

Glutaredoxin-dependent peroxiredoxin from poplar: protein-protein interaction and catalytic mechanism

Recently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained approximately 25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx.     

Role of vicinal cysteine pairs in metalloid sensing by the ArsD As(III)-responsive repressor

ArsD is a 120-residue repressor that regulates expression of the arsRDABC arsenical resistance operon of plasmid R773 in Escherichia coli. ArsD is released from arsRDABC promoter DNA by binding of the compounds with the metalloids As(III) or Sb(III). ArsD has three vicinal cysteine pairs, Cys-12 and Cys-13, Cys-112 and Cys-113 and Cys-119 and Cys-120. In this study, the role of these three cysteine pairs was investigated. Mutation or deletion of Cys-119-Cys-120 had no effect on repression or metalloid responsiveness in vivo or in vitro. Mutagenesis of either the Cys-12-Cys-13 pair or the Cys-112-Cys-113 pair had no effect on repression but produced loss of inducibility, suggesting that both Cys-12-Cys-13 and Cys-112-Cys-113 may be required for As(III) or Sb(III) responsiveness. Assays of binding of wild-type and mutant ArsDs by As(III) affinity chromatography showed that each of the three vicinal cysteine pairs is capable of binding As(III) independently. The effect of As(III) or Sb(III) on intrinsic protein fluorescence was used to examine the properties of individual cysteine pairs. The fluorescence of Trp-97 was shown to be quenched by the addition of Sb(III) or As(III). The vicinal Cys-112-Cys-113 pair was required for the majority of the metalloid-dependent quenching of Trp-97 fluorescence. The data are consistent with a model in which Cys-12-Cys-13 and Cys-112-Cys-113 form independent As(III) binding sites, both of which are required for in vivo ArsD function.