Active Site Cysteine Residue Research Articles

Molecular cloning and characterization of a cathepsin B gene from the Chinese shrimp Fenneropenaeus chinensis

Cathepsin B is a unique member of the cathepsin superfamily, which acts as both an endopeptidase and peptidyl-dipeptidase. To obtain a better understanding of this enzyme, we cloned a cDNA encoding cathepsin B from the muscle of Fenneropenaeus chinensis (FcCB). FcCB contained a 996-bp open reading frame (ORF) encoding a protein of 331 amino acid residues with a putative signal peptide and a propeptide_C1 at the N-terminal, a glutamine oxyanion hole and active site cysteine, histidine and asparagine residues. A region from residue 79 to 327 conferred the peptidase activity of FcCB. Pair-wise and multiple sequence alignment with 17 other organisms, including ten different vertebrate species, five different invertebrate species and two different plant species, indicated that the signal peptide and the propeptide_C1 at the N-terminal of FcCB were less conserved than the mature protein, except when compared with Penaeus monodon, Litopenaeus vannamei and Marsupenaeus japonicas, all of which belong to the genus Penaeus. The expression of FcCB in the hepatopancreas was higher than that in the gill. The expression of FcCB in the gill was higher than that in the muscle. A challenge test was performed to reveal the responses of FcCB in different tissues to white spot syndrome virus (WSSV) infection, which causes serious economic losses in the shrimp farming industry. The FcCB gene expressions in the ectoderm, mesoderm and entoderm were not the same prior to WSSV infection, but at 6 h after WSSV challenge, the FcCB expression in the gill, hepatopancreas and muscle was up-regulated, suggesting that FcCB might be involved in the immune response to WSSV. Three single nucleotide polymorphisms (SNPs) were identified in the FcCB gene, involving C/T transitions, which are known as mutation hot spots. Notably, the three SNPs constituted a haplotype that can be used as an indicator of the haplotype block. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps, were obtained using a high-resolution melting (HRM) method. Associated factors, including observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF) and P-values for the deviation from Hardy–Weinberg equilibrium (HWE), were obtained. For the association analysis with WSSV resistance, the P-values were calculated using Pearson's chi-square test. In the two groups, the MAFs of all sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distribution of the C-984T mutation site between the two groups was not significantly different. These results lead to a better understanding of the molecular mechanisms of the host–virus interaction and provide useful information for solving the WSSV problem.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

Targeting the Arginine Phosphatase YwlE with a Catalytic Redox-Based Inhibitor

Protein phosphatases are critical regulators of cellular signaling in both eukaryotes and prokaryotes. The majority of protein phosphatases dephosphorylate phosphoserine/phosphothreonine or phosphotyrosine residues. Recently, however, YwlE, a member of the low-molecular weight protein tyrosine phosphatase (LMW-PTP) family, was shown to efficiently target phosphoarginine. YwlE shares several sequence motifs with this family including the C(X)4 CR(S/T) motif that is crucial for catalysis and redox regulation of the enzyme. Herein we confirm that Cys9 and Cys14 play important roles in YwlE catalysis and regulation. On the basis of these observations, we designed and synthesized a YwlE inhibitor, denoted cyc-SeCN-amidine, that irreversibly inhibits YwlE (kinact/KI = 310 M(-1) min(-1)) by inducing disulfide bond formation between the two active site cysteine residues. Interestingly, inactivation appears to be catalytic, since the compound is neither destroyed nor altered after enzyme inhibition. Although the exact mechanism of disulfide induction remains elusive, we propose several potential mechanisms accounting for the cyc-SeCN-amidine mediated inhibition of YwlE. These findings could stimulate the design of similar selenium-based compounds targeting other redox-sensitive enzymes.

Kinetics of O6-Pyridyloxobutyl-2′-deoxyguanosine Repair by Human O6-alkylguanine DNA Alkyltransferase

Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine (O(6)-POB-dG) lesions. If not repaired, O(6)-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that O(6)-POB-dG can be directly repaired by O(6)-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from O(6)-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of O(6)-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific O(6)-POB-dG adducts within K-ras and p53 gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI(+)-MS/MS analysis of O(6)-POB-dG remaining in DNA over time. We found that the second-order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences) but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of O(6)-POB-dG was 2-7 times slower than that of O(6)-Me-dG adducts. To evaluate the contribution of AGT to O(6)-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and O(6)-POB-dG adduct repair over time was monitored by HPLC-ESI(+)-MS/MS. We found that HBEC cells were capable of removing O(6)-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (O(6)-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of O(6)-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer.

90-kDa ribosomal S6 kinase 1 is inhibited by S-glutathionylation of its active-site cysteine residue during oxidative stress

Previously, we reported that p90-RSK1 phosphorylates neuronal nitric oxide synthase (nNOS) at Ser847 in cells treated with mitogens, leading to the inhibition of NOS activity. Here, we show RSK1 Cys223 glutathionylation limits the activity of the enzyme following an oxidative stimulus and attenuates the nNOS phosphorylation. Treatment of RSK1 with diamide/glutathione results in inactivation of the enzyme in vitro. Mutagenesis studies confirmed that S-glutathionylation of Cys223 is both necessary and sufficient for this inhibition of RSK1. In transfected cells expressing RSK1 and nNOS, treatment with diamide caused a decrease in EGF-induced phosphorylation of nNOS at Ser847. Cells expressing mutant RSK1 (C223S) proved resistant in this regard. Thus, RSK1 Cys223 glutathionylation may contribute to regulate the levels of NO in the brain.

P74: Novel human mitochondrial enzyme can transform nitrite into nitric oxide

Endogenous metabolism of nitrate to nitrite to nitric oxide (NO) affects numerous biological events in humans, such as blood pressure control and hypoxic vasodilation, yet the molecular mechanism of this transformation remains uncertain and controversial. Nitrate and nitrite conversion to NO are reductive processes, requiring electron and proton transfer reactions, suggesting that oxidoreductase enzymes are involved. Nitrate reduction may occur through a combination of endogenous (i.e. human enzymes) and exogenous (i.e. commensal bacteria) nitrate reductase enzymes, while nitrite reduction to nitric oxide is likely catalyzed by endogenous nitrite reductase enzymes. Several mechanisms have been investigated, yet no conclusive data are available to support a single enzyme as an indispensable component for reduction of nitrite to NO in human tissue. It is likely that different enzyme systems are active in specific tissues under differential hypoxia, pH and co-factor regulatory control. We have identified a novel human nitrite reductase enzyme, which we hypothesize may contribute to mitochondrial reduction of nitrite to NO. The mitochondria amidoxime reducing component (mARC) enzyme is widely expressed in human tissue, but has an undefined physiological function. We hypothesize that mARC catalyzes the NADH-dependent reduction of nitrite to NO. To test this hypothesis the kinetics of nitrite reduction to NO by human mARC was investigated. Recombinant mARC was generated using standard molecular biology techniques, and then isolated using metal affinity chromatography. Site directed mutagenesis was used to change the active site cysteine into alanine. Human cytochrome b5 and cytochrome b5 reductase were also isolated to determine if mARC can utilize NADH as an electron source in conjunction with these enzymes. Nitric oxide chemiluminescence spectroscopy was used to measure NO-formation rates under anaerobic conditions. Our study established that mARC can generate NO from nitrite in the presence of NADH, cytochrome b5, and cytochrome b5 reductase at pH 7.4. The maximum velocity (Vmax) of NO-formation measured was 5 nmoles NO s−1 mg−1 protein. Moreover, mutation of the putative active site cysteine residue to alanine, and substitution of tungsten for molybdenum, completely abolished enzyme activity. The kinetic data supports our hypothesis and establishes that human mARC is capable of catalyzing reduction of nitrite to NO. Moreover, these data suggest that cysteine 270 and molybdenum are important in the transformation of nitrite to NO. Disclosure Supported by institution training grant (T32).

Identification of Novel Irreversible Inhibitors of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Haemophilus influenzae

Uridinediphospho-N-acetylglucosamine enolpyruvyl transferase (MurA, E.C. 2.5.1.7) is an essential bacterial enzyme that catalyzes the first step of the cell wall biosynthetic pathway, which involves the transfer of an enolpyruvyl group from phosphoenolpyruvate to uridinediphospho-Nacetylglucosamine. In this study, novel inhibitors of Haemophilus influenzae MurA (Hi MurA) were identified using high-throughput screening of a chemical library from the Korea Chemical Bank. The identified compounds contain a quinoline moiety and have much lower effective inhibitory concentrations (IC(50)) than fosfomycin, a wellknown inhibitor of MurA. These inhibitors appear to covalently modify the sulfhydryl group of the active site cysteine (C117), since the C117D mutant Hi MurA was not inhibited by these compounds and excess dithiothreitol abolished their inhibitory activities. The increased mass value of Hi MurA after treatment with the identified inhibitor further confirmed that the active-site cysteine residue of Hi MurA is covalently modified by the inhibitor.

Inhibitory zinc sites in enzymes

Several pathways increase the concentrations of cellular free zinc(II) ions. Such fluctuations suggest that zinc(II) ions are signalling ions used for the regulation of proteins. One function is the inhibition of enzymes. It is quite common that enzymes bind zinc(II) ions with micro- or nanomolar affinities in their active sites that contain catalytic dyads or triads with a combination of glutamate (aspartate), histidine and cysteine residues, which are all typical zinc-binding ligands. However, for such binding to be physiologically significant, the binding constants must be compatible with the cellular availability of zinc(II) ions. The affinity of inhibitory zinc(II) ions for receptor protein tyrosine phosphatase β is particularly high (K i = 21 pM, pH 7.4), indicating that some enzymes bind zinc almost as strongly as zinc metalloenzymes. The competitive pattern of zinc inhibition for this phosphatase implicates its active site cysteine and nearby residues in the coordination of zinc. Quantitative biophysical data on both affinities of proteins for zinc and cellular zinc(II) ion concentrations provide the basis for examining the physiological significance of inhibitory zinc-binding sites in proteins and the role of zinc(II) ions in cellular signalling. Regulatory functions of zinc(II) ions add a significant level of complexity to biological control of metabolism and signal transduction and embody a new paradigm for the role of transition metal ions in cell biology.

On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

The nitrosated bile acid DNA lesion O6 -carboxymethylguanine is a substrate for the human DNA repair protein O6 -methylguanine-DNA methyltransferase

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O6-carboxymethylguanine (O6-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O6-CMG is not a substrate for the human version of the DNA damage reversal protein O6-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O6-alkylguanine lesions by removing alkyl groups from the O6-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O6-methylguanine (O6-MeG) or O6-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O6-alkyl group and its transfer to the active-site cysteine residue of MGMT. O6-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O6-CMG is a potential causative agent.

Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57’s active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H2O2 inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).

Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.

Rapid Equilibrium Kinetic Analysis of Arsenite Methylation Catalyzed by Recombinant Human Arsenic (+3 Oxidation State) Methyltransferase (hAS3MT)

In the human body, arsenic is metabolized by methylation. Understanding this process is important and provides insight into the relationship between arsenic and its related diseases. We used the rapid equilibrium kinetic model to study the reaction sequence of arsenite methylation. The results suggest that the mechanism for arsenite methylation is a completely ordered mechanism that is also of general interest in reaction systems with different reductants, such as tris(2-carboxyethyl)phosphine, cysteine, and glutathione. In the reaction, cysteine residues of recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. S-Adenosyl-l-methionine (AdoMet) is the first-order reactant, which modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. As the second-order reactant, reductant reduces the disulfide bond, most likely between Cys-250 and another cysteine residue of hAS3MT, and exposes the active site cysteine residues for binding trivalent inorganic arsenic (iAs(3+)) to give monomethylarsonic dicysteine (MADC(3+)). In addition, the reaction can be extended to further methylate MADC(3+) to dimethylarsinic cysteine (DAMC(3+)). In the methylation reaction, the β-pleated sheet content of hAS3MT is increased, and the hydrophobicity of the microenvironment around the active sites is decreased. Similarly, we confirm that both the high β-pleated sheet content of hAS3MT and the high dissociation ability of the enzyme-AdoMet-reductant improve the yield of dimethylated arsenicals.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.

Cyanamide-mediated Inhibition of N-acetyltransferase 1

Cyanamide has been used for decades for medical intentions in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolized in vivo mainly via coenzyme A dependent N-acetylation by N-acetyltransferases. Although described to be a substrate for N-acetyltransferases (NATs), cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for N-acetyltransferases. Therefore, a more detailed investigation of its interrelations with N-acetyltransferases was performed. We analyzed the impact of cyanamide on NAT1 activities of human monocytes (monocytic THP-1 cells) using the classical substrate p-aminobenzoic acid. We found that a 24h treatment with physiologically relevant concentrations of cyanamide decreased the NAT1 activity significantly. Based on this observation we performed additional experiments using recombinant human NAT1 and NAT2 to achieve further insights. In detail a significant dose- and time-dependent inhibition of NAT1 activity was observed for 100 and 1000μM cyanamide using recombinant human NAT1*4. However, cyanamide did not inhibit recombinant NAT2*4. Experiments testing cyanamide as substrate did not provide evidence that cyanamide is metabolized via coenzyme A dependent N-acetylation in vitro by human NAT1 or NAT2, THP-1 or human liver cytosol. Therefore we can conclude that the observed enzyme inhibition (around 50% and 25% after treatment with 0.5 and 0.25mM CA, respectively) is not based on substrate-dependent down-regulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition (significant inhibition after 30min and 2h for 1000 and 100μM CA, respectively). Addition of the reduction agent dithiothreitol (DTT) did not modify the effect, indicating that oxidative processes that can be reversed by 5mM DTT are not likely involved in the inhibition. Taken together our results show that cyanamide is able to inhibit NAT1 most likely via interaction with the active site cysteine residue. Thereby cyanamide might modulate NAT1 dependent detoxification and activation of arylamines.

An unusual tandem‐domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene. The peculiarity of the PhsE protein is its domain architecture which is constituted of two rhodanese domains each with an active site cysteine. The N-terminal rhodanese domain is preceded by a lipoprotein signal peptide anchoring PhsE on the outside of the cytoplasmic membrane. In vitro sulfur-transferase activity of recombinant PhsE variants was measured for both domains contrasting with other tandem-domain rhodaneses in which usually only the C-terminal domain has been found to be active. The genetic context of phsE shows that it is part of a six-gene operon displaying homology with gene clusters encoding respiratory molybdoenzymes of the PhsA/PsrA family, possibly involved in the reduction of sulfur compounds. Our data suggest, however, that the presence of sulfide in the medium is responsible for the high expression of PhsE in Desulfitobacterium, where it could play a role in the sulfur homeostasis of the cell.

Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAsIII) and its intermediate metabolites such as monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMAIII and DMAIII) but not by iAsIII. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMAIII directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation.

Reversible inactivation of Ca2+/calmodulin-dependent protein kinase IV by oxidation of the active-site cysteine residue

Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) is serine/threonine-specific protein kinase that is involved in many signaling cascades, and plays a key role in memory. CaMKIV is activated via binding to Ca 2+ /CaM complex and phosphorylation by upstream kinase, CaM kinase. In the present study, we investigate direct mechanism of inactivation of CaMKIV by oxidative stress. We show that CaMKIV is directly inhibited by its sulfenic acid formation at the Cys198. In situ studies demonstrated that treatment of CaMKIV with hydrogen peroxide (H 2 O 2 ) results in inactivation of the enzyme, with a concomitant sulfenic acid formation of CaMKIV at Cys198. The methodology of detection of sulfenic acid formation is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Mutagenesis studies confirmed that sulfenic acid formation of Cys198 is both necessary and sufficient for the inhibition of CaMKIV by H 2 O 2 . In transfected cells expressing CaMKIV, treatment with H 2 O 2 caused a reversible decrease in CaMKIV activity. Cells expressing mutant CaMKIV (C198V) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKIV via its sulfenic acid formation at Cys198 is an important mechanism in processing calcium signal transduction in cells.

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.