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51.
Efrén Ordó?ez Karolien Van Belle Goedele Roos Sandra De Galan Michal Letek Jose A. Gil Lode Wyns Luis M. Mateos Joris Messens 《The Journal of biological chemistry》2009,284(22):15107-15116
We identified the first enzymes that use mycothiol and mycoredoxin in a
thiol/disulfide redox cascade. The enzymes are two arsenate reductases from
Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key
role in the defense against arsenate. In vivo knockouts showed that
the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol
biosynthesis pathway confer arsenate resistance. With steady-state kinetics,
arsenite analysis, and theoretical reactivity analysis, we unraveled the
catalytic mechanism for the reduction of arsenate to arsenite in C.
glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct
formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for
which the function was never shown before, reduces the thiol-arseno bond and
forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule
of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is
reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low
specificity constant of ∼5 m-1 s-1,
typically for a thiol/disulfide cascade with nucleophiles on three different
molecules. With the in vitro reconstitution of this novel electron
transfer pathway, we have paved the way for the study of redox mechanisms in
actinobacteria.The frequent abundance of arsenic in the environment has guided the
evolution of enzymes for the reduction of arsenate
(As(V))4
(1). Arsenate reductases
(ArsCs) are unusual among well studied enzyme classes, because there is not a
single family of evolutionarily related sequences. The structural folds and
mechanisms that they are using are fundamentally different and arose
independently during evolution
(2). Arsenate reductases are
small cytoplasmic redox enzymes that reduce arsenate to arsenite (As(III)) by
the sequential involvement of three different thiolate nucleophiles that
function as a redox cascade. As such, arsenate reductases from different
organisms often work together with the thiol/disulfide mechanism in the
cell.The major and most ubiquitous system for protection against oxidative
stress and to maintain the intracellular thiol homeostasis is the thioredoxin
system that is composed of Trx (thioredoxin) and TrxR (thioredoxin reductase)
(3). In addition to the
thioredoxin system, most living organisms contain low molecular weight thiol
compounds that serve as a buffer to avert disulfide stress. In eukaryotes and
Gram-negative bacteria, the redox level is maintained by redox cycling of
glutathione (GSH) with Grx (glutaredoxin) and glutathione reductase
(4). Gram-positive bacteria,
like Staphylococcus aureus, produce no glutathione, but millimolar
quantities of reduced coenzyme A is the predominant thiol, which is kept
reduced with a NADPH-dependent flavoenzyme, coenzyme A disulfide reductase
(5). Also actinobacteria, like
Corynebacterium glutamicum, produce no GSH, but instead they contain
millimolar concentrations of MSH (mycothiol; chemically
1D-myo-inosityl-2-[N-acetyl-l-cysteinyl]
amido-2-deoxy-α-d-glucopyranoside), a pseudodisaccharide
containing a cysteine moiety as a reactive thiol
(6). Its oxidized form is
mycothione (MSSM). In actinobacteria, MTR (mycothione reductase) is the
NADPH-dependent flavoenzyme that reduces MSSM in order to maintain the
intracellular redox homeostasis to allow the proper functioning of a variety
of biological functions
(7).Arsenate reductases are part of a defense mechanism of the cell against
toxic arsenate. Their genes are most of the time found in an operon together
with arsenite sensing and efflux genes
(8). Based on the mechanism
used to reduce arsenate to arsenite, two distinct ArsC classes can be defined.
The first one is the thioredoxin-coupled ArsC class represented by S.
aureus pI258 ArsC and Bacillus subtilis ArsC
(9–11).
Both enzymes use the structural fold of low molecular weight tyrosine
phosphatase and need Trx to start a second catalytic cycle
(12–14).
The second class is the GSH/glutaredoxin-coupled class represented by
Escherichia coli plasmid R773 ArsC
(15,
16), the eukaryotic Acr2p
reductase from Saccharomyces cerevisiae
(17), and ArsC from
Leishmania major
(18). In this second class,
two different structural folds are found; E. coli R773 ArsC partially
resembles glutaredoxin (19),
whereas the eukaryotic ArsCs have a rhodanese fold like the Cdc25a cell cycle
control phosphatase (20).
Notably, all arsenate reductases have a thiolate nucleophile at the N-terminal
end of an α-helix. The active site of the ArsCs with a phosphatase-like
scaffold is conserved (root mean square deviation of 0.54 Å) with a
catalytically important Arg on position Cys+6.In C. glutamicum, there are four arsC genes located on
different places in the chromosome
(21): one orphan arsC
gene (arsC4) and three arsC genes
(arsC1-arsC1′ and arsC2) present in two
ars operons. We show here that two of the encoded proteins, Cg_ArsC1
and Cg_ArsC2 (with 66% sequence identity) are members of a new third class,
the mycothiol- and mycoredoxin-dependent arsenate reductases. Both the genes
of arsC1 and arsC2, together with the genes for the enzymes
of the mycothiol biosynthesis pathway are involved in arsenate resistance in
C. glutamicum. We have reconstituted in vitro a novel
electron transfer network containing, next to Cg_ArsC1 or Cg_ArsC2, mycothiol,
mycoredoxin, and mycothione reductase. As such, the mechanism for the
reduction of arsenate by C. glutamicum could be unraveled. 相似文献
52.
53.
Cultivation techniques were used to study the heterotrophic bacterial diversity in two microbial mat samples originating from
the littoral zone of two continental Antarctic lakes (Forlidas Pond and Lundstr?m Lake) in the Dufek Massif (within the Pensacola
Mountains group of the Transantarctic Mountains) and Shackleton Range, respectively. Nearly 800 isolates were picked after
incubation on several growth media at different temperatures. They were grouped using a whole-genome fingerprinting technique,
repetitive element palindromic PCR and partial 16S rRNA gene sequencing. Phylogenetic analysis of the complete 16S rRNA gene
sequences of 82 representatives showed that the isolates belonged to four major phylogenetic groups: Actinobacteria, Bacteroidetes, Proteobacteria and Firmicutes. A relatively large difference between the samples was apparent. Forlidas Pond is a completely frozen water body underlain
by hypersaline brine, with summer thaw forming a slightly saline littoral moat. This was reflected in the bacterial diversity
with a dominance of isolates belonging to Firmicutes, whereas isolates from the freshwater Lundstr?m Lake revealed a dominance of Actinobacteria. A total of 42 different genera were recovered, including first records from Antarctica for Albidiferax, Bosea, Curvibacter, Luteimonas, Ornithinibacillus, Pseudoxanthomonas, Sphingopyxis and Spirosoma. Additionally, a considerable number of potential new species and new genera were recovered distributed over different phylogenetic
groups. For several species where previously only the type strain was available in cultivation, we report additional strains.
Comparison with public databases showed that overall, 72% of the phylotypes are cosmopolitan whereas 23% are currently only
known from Antarctica. However, for the Bacteroidetes, the majority of the phylotypes recovered are at present known only from Antarctica and many of these represent previously
unknown species. 相似文献
54.
Villadangos AF Van Belle K Wahni K Dufe VT Freitas S Nur H De Galan S Gil JA Collet JF Mateos LM Messens J 《Molecular microbiology》2011,82(4):998-1014
Arsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k(cat)/K(M) value which is 10(3) times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface. 相似文献
55.
Huang C Stakenborg T Cheng Y Colle F Steylaerts T Jans K Van Dorpe P Lagae L 《Biosensors & bioelectronics》2011,26(7):3121-3126
In this report, we demonstrate a label-free genosensor based on DNA hairpins coupled to gold coated sensor surfaces. The hairpin probes were labeled with a thiolated moiety for immobilization at the 5' end and with a fluorophore for signal transduction at the 3' end. In the absence of the complement, the fluorophore is quenched by energy transfer to the gold surface. Addition of the target sequence leads to the hairpin unfolding, and releases the fluorescent signal. This built-in property, using a gold film as both the immobilizing substrate and quenching agent, has the advantage of simplicity in design and ease of further integration. Our results showed that lengths of both the stem and the loop structures have significant effects on the sensor performance. Hybridization kinetics was investigated for various probe/target lengths and concentrations. An optimized hairpin probe gave a fluorescent signal increase of 39 folds after hybridization, which is much higher than the earlier reported results. A limit of detection (LOD) down to 0.3 nM for the complementary target DNA detection has been achieved. The developed sensor was further successfully applied for the detection of single-base mismatch targets, as well as for the direct detection of PCR products. 相似文献
56.
Jaspers K Versluis B Leiner T Dijkstra P Oostendorp M van Golde JM Post MJ Backes WH 《PloS one》2011,6(1):e16159
The objective of this study was to compare the blood pool agent Gadomer with a small contrast agent for the visualization of ultra-small, collateral arteries (diameter<1 mm) with high resolution steady-state MR angiography (SS-MRA) in a rabbit hind limb ischemia model. Ten rabbits underwent unilateral femoral artery ligation. On days 14 and 21, high resolution SS-MRA (voxel size 0.49×0.49×0.50 mm3) was performed on a 3 Tesla clinical system after administration of either Gadomer (dose: 0.10 mmol/kg) or a small contrast agent (gadopentetate dimeglumine (Gd-DTPA), dose: 0.20 mmol/kg). All animals received both contrast agents on separate days. Selective intra-arterial x-ray angiograms (XRAs) were obtained in the ligated limb as a reference. The number of collaterals was counted by two independent observers. Image quality was evaluated with the contrast-to-noise ratio (CNR) in the femoral artery and collateral arteries. CNR for Gadomer was higher in both the femoral artery (Gadomer: 73±5 (mean ± SE); Gd-DTPA: 40±3; p<0.01) and collateral arteries (Gadomer: 18±4; Gd-DTPA: 9±1; p = 0.04). Neither day of acquisition nor contrast agent used influenced the number of identified collateral arteries (p = 0.30 and p = 0.14, respectively). An average of 4.5±1.0 (day 14, mean ± SD) and 5.3±1.2 (day 21) collaterals was found, which was comparable to XRA (5.6±1.7, averaged over days 14 and 21; p>0.10). Inter-observer variation was 24% and 18% for Gadomer and Gd-DTPA, respectively. In conclusion, blood pool agent Gadomer improved vessel conspicuity compared to Gd-DTPA. Steady-state MRA can be considered as an excellent non-invasive alternative to intra-arterial XRA for the visualization of ultra-small collateral arteries. 相似文献
57.
Joris Messens José C. Martins Elke Brosens Karolien Van Belle Doris M. Jacobs Rudolph Willem Lode Wyns 《Journal of biological inorganic chemistry》2002,7(1-2):146-156
Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM). 相似文献
58.
An amphiphilic silk-like protein polymer was efficiently produced in the yeast Pichia pastoris. The secreted product was fully intact and was purified by solubilization in formic acid and subsequent precipitation of denatured host proteins upon dilution with water. In aqueous alkaline solution, the negatively charged acidic polymer assumed extended helical (silk III-like) and unordered conformations. Upon subsequent drying, it assumed a conformation rich in beta-turns. In water at low pH, the uncharged polymer aggregated and the solution became turbid. Concentrated solutions in 70% (v/v) formic acid slowly formed gels. Replacement of the formic acid-water mixture with methanol and subsequent drying resulted in beta-sheets, which stacked into fibril-like structures. The novel polymer instantaneously lowered the air-water interfacial tension under neutral to alkaline conditions and reversed the polarity of hydrophobic and hydrophilic solid surfaces upon adsorption. 相似文献
59.
Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation. A screening method for rapidly determining said substrate selectivity was developed. Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant. A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX. A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate. A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities. 相似文献
60.
Marcoen A van Cotthem K Billiet K Beyers W 《Tijdschrift voor gerontologie en geriatrie》2002,33(4):156-165
Subjective well-being is an important and relevant theme in psychogerontological theory and practice. Against the background of the distinction between hedonistic and eudaimonic conceptions of well-being a six-dimensional relational model of subjective well-being is described. A new instrument for the measurement of psychological, physical, social, material, cultural and existential well-being was tested in a sample of 366 elderly persons (61% women) aged 60 years and older. After item analysis and exploratory factor analysis of the different scales, 56 items of the original 72 were retained for six more homogeneous scales with acceptable Cronbach's alphas between .89 en .61. Relations were found between the scores on different scales--especially on the scales for Physical, Psychological, Existential and Cultural Well-being--and some demographic and person-related variables such as, number of children, presence or absence of daughters, living independently, availability of friends, etc. 相似文献