Cysteine Residues Cys Research Articles

FGE‐sulfatase‐based bacterial biosensor with single copy evolved sensing cassette for arsenic detection

AbstractBACKGROUNDArsenic (As) is classified as a Group 1 human carcinogen, and the maximum arsenic contamination level allowed in drinking water is10 μg L−1. In this study, a genetic‐based whole‐cell biosensor for arsenic detection was constructed and engineered for rapid and more efficient arsenic detection.RESULTSThe promoter and repressor responsive to arsenic (ArsBR) were newly isolated from Bacillus cereus ATCC14579. The reporter gene was atsBA carrying formylglycine‐generating enzyme (FGE) and sulfatase, which provided a fluorescent product after reacting with 4‐methylumbelliferyl sulfate (4‐MUS). To improve the performance of the arsenic biosensor, firstly, more efficient Escherichia coli host strains were constructed, including the disruption of the arsenic efflux pump, multidrug efflux pump and arsenical‐resistant genes; secondly, a repressor (ArsBR) was engineered by an error‐prone polymerase chain reaction (PCR) technique, aiming to lower the detection limit; lastly, a single copy biosensor cassette in an E. coli chromosome was constructed.CONCLUSIONThe stable and sensitive biosensor was able to detect As(III) at 5 μg L−1 and As(V) at 50 μg L−1 in 3.5 h with a very low fluorescent background. Moreover, the roles of all seven cysteine residues of the repressor were also investigated. Interestingly, the six cysteine residues Cys 28, 34, 36, 89, 90 and 100 are functionally involved in arsenic sensing, but not Cys 101. © 2019 Society of Chemical Industry

Spectral characteristics of the mutant form GGBP/H152C of D-glucose/D-galactose-binding protein labeled with fluorescent dye BADAN: influence of external factors

The mutant form GGBP/H152C of the D-glucose/D-galactose-binding protein with the solvatochromic dye BADAN linked to cysteine residue Cys 152 can be used as a potential base for a sensitive element of glucose biosensor system. We investigated the influence of various external factors on the physical-chemical properties of GGBP/H152C-BADAN and its complex with glucose. The high affinity (Kd = 8.5 µM) and high binding rate of glucose make GGBP/H152C-BADAN a good candidate to determine the sugar content in biological fluids extracted using transdermal techniques. It was shown that changes in the ionic strength and pH of solution within the physiological range did not have a significant influence on the fluorescent characteristics of GGBP/H152C-BADAN. The mutant form GGBP/H152C has relatively low resistance to denaturation action of GdnHCl and urea. This result emphasizes the need to find more stable proteins for the creation of a sensitive element for a glucose biosensor system.

Activation of oligopeptidase B from Streptomyces griseus by thiol-reacting reagents is independent of the single reactive cysteine residue

Oligopeptidase B from Streptomyces griseus was cloned and characterized to clarify the substrate recognition mechanism and the role of a reactive cysteine residue in family S9 prolyl oligopeptidases (POPs). The cloned enzyme, SGR-OpdB, was annotated as a putative family S9 prolyl oligopeptidase based on its deduced amino acid sequence, in which a sole cysteine residue Cys 544 is present close to the catalytic Asp residue in the C-terminal region. The protein was identified as oligopeptidase B, a member of the subfamily S9a of the family S9 POPs, as judged by its substrate specificity and enzymatic characteristics. Its enzymatic activity was markedly enhanced by high NaCl concentration and the reducing reagents dithiothreitol (DTT) and reduced glutathione (GSH). It is particularly interesting that oxidized glutathione (GSSG) also enhanced SGR-OpdB activity. The SGR-OpdB C544A mutant was constructed and characterized to clarify the role of the putative reactive Cys residue, Cys 544. Surprisingly, the enzymatic activity of the Cys-free mutant was also markedly activated by the general thiol-reacting reagent DTT, GSH, and GSSG. To our knowledge, this is the first report of activity-enhancing effects of thiol-reacting reagents toward Cys-free enzymes. Results clarified the role of additives in inducing conformational change of SGR-OpdB into active peptidase.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys 189 is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from PII SVMPs. The ECD motif is stabilized by the Cys 277–Cys 310 disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu 276 of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other PIII SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other PIII members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain.

ERp16, an Endoplasmic Reticulum-resident Thiol-disulfide Oxidoreductase: BIOCHEMICAL PROPERTIES AND ROLE IN APOPTOSIS INDUCED BY ENDOPLASMIC RETICULUM STRESS

We have characterized the properties and putative role of a mammalian thioredoxin-like protein, ERp16 (previously designated ERp18, ERp19, or hTLP19). The predicted amino acid sequence of the 172-residue human protein contains an NH(2)-terminal signal peptide, a thioredoxin-like domain with an active site motif (CGAC), and a COOH-terminal endoplasmic reticulum (ER) retention sequence (EDEL). Analyses indicated that the mature protein (comprising 146 residues) is generated by cleavage of the 26-residue signal peptide and is localized in the lumen of the ER. Biochemical experiments with the recombinant mature protein revealed it to be a thioldisulfide oxidoreductase. Its redox potential was about -165 mV; its active site cysteine residue Cys(66) was nucleophilic with a pK(a) value of approximately 6.6; it catalyzed the formation, reduction, and isomerization of disulfide bonds, with the unusual CGAC active site motif being responsible for these activities; and it existed as a dimer and underwent a redox-dependent conformational change. The observations that the redox potential of ERp16 (-165 mV) was within the range of that of the ER (-135 to -185 mV) and that ERp16 catalyzed disulfide isomerization of scrambled ribonuclease A suggest a role for ERp16 in protein disulfide isomerization in the ER. Expression of ERp16 in HeLa cells inhibited the induction of apoptosis by agents that elicit ER stress, including brefeldin A, tunicamycin, and dithiothreitol. In contrast, expression of a catalytically inactive mutant of ERp16 potentiated such apoptosis, as did depletion of ERp16 by RNA interference. Our results suggest that ERp16 mediates disulfide bond formation in the ER and plays an important role in cellular defense against prolonged ER stress.

A Vertebrate-type Ferredoxin Domain in the Na+-translocating NADH Dehydrogenase from Vibrio cholerae

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mössbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys(70), Cys(76), Cys(79), and Cys(111). Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.

Oxygen-independent Coproporphyrinogen-III Oxidase HemN from Escherichia coli

In bacteria the oxygen-independent coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of coproporphyrinogen-III to protoporphyrinogen-IX. The Escherichia coli hemN gene encoding a putative part of this enzyme was overexpressed in E. coli. Anaerobically purified HemN is a monomeric protein with a native M(r) = 52,000 +/- 5,000. A newly established anaerobic enzyme assay was used to demonstrate for the first time in vitro coproporphyrinogen-III oxidase activity for recombinant purified HemN. The enzyme requires S-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis. An oxygen-sensitive iron-sulfur cluster was identified by absorption spectroscopy and iron analysis. Cysteine residues Cys(62), Cys(66), and Cys(69), which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function. Completely conserved residues Tyr(56) and His(58), localized closely to the cysteine-rich motif, were found to be important for iron-sulfur cluster integrity. Mutation of Gly(111) and Gly(113), which are part of the potential GGGTP S-adenosyl-l-methionine binding motif, completely abolished enzymatic function. Observed functional properties in combination with a recently published computer-based enzyme classification (Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001) Nucleic Acids Res. 29, 1097-1106) identifies HemN as "Radical SAM enzyme." An appropriate enzymatic mechanism is suggested.

Construction and Characterization of a Monomeric Insulin Receptor

A mutant insulin receptor was constructed by replacing cysteine residues Cys(524), Cys(682), Cys(683), and Cys(685) with serine. The mutant was expressed in COS7 and Chinese hamster ovary cells, did not form covalently linked dimers, and was present at the cell surface. There was half as much insulin binding activity at the cell surface in cells expressing the mutant compared with that in cells expressing the wild type receptor. The intracellular processing of the mutant receptor was affected, since its beta-subunit migrated more slowly than that of the wild type receptor on SDS-PAGE. The mutant was capable of insulin-dependent autophosphorylation and phosphorylation of insulin receptor substrate-1 in vivo and could be cross-linked into receptor dimers when membrane-bound. The amount of insulin-dependent autophosphorylation of the mutant receptor was half that of the wild type receptor. However, after solubilization the monomeric insulin receptor had minimal autophosphorylation activity, and, unlike the naturally occurring monomeric receptor tyrosine kinases, the solubilized monomeric insulin receptor did not dimerize in response to insulin binding as determined by sucrose density gradient centrifugation.

Mapping of a hypovirus p29 protease symptom determinant domain with sequence similarity to potyvirus HC-Pro protease.

Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica results in a spectrum of phenotypic changes that can include alterations in colony morphology and significant reductions in pigmentation, asexual sporulation, and virulence (hypovirulence). Deletion of 88% [Phe(25) to Pro(243)] of the virus-encoded papain-like protease, p29, in the context of an infectious cDNA clone of the prototypic hypovirus CHV1-EP713 (recombinant virus Deltap29) partially relieved virus-mediated suppression of pigmentation and sporulation without altering the level of hypovirulence. We now report mapping of the p29 symptom determinant domain to a region extending from Phe(25) through Gln(73) by a gain-of-function analysis following progressive repair of the Deltap29 deletion mutant. This domain was previously shown to share sequence similarity [including conserved cysteine residues Cys(38), Cys(48), Cys(70), and Cys(72)] with the N-terminal portion of the potyvirus-encoded helper component-proteinase (HC-Pro), a multifunctional protein implicated in aphid-mediated transmission, genome amplification, polyprotein processing, long-distance movement, and suppression of posttranscriptional silencing. Substitution of a glycine residue for either Cys(38) or Cys(48) resulted in no qualitative or quantitative changes in virus-mediated symptoms. Unexpectedly, mutation of Cys(70) resulted in a very severe phenotype that included significantly reduced mycelial growth and profoundly altered colony morphology. In contrast, substitution for Cys(72) resulted in a less severe symptom phenotype approaching that observed for Deltap29. The finding that p29-mediated symptom expression is influenced by two cysteine residues that are conserved in the potyvirus-encoded HC-Pro raises the possibility that these related viral-papain-like proteases function in their respective fungal and plant hosts by impacting ancestrally related regulatory pathways.