Biomedical subjects

A Rietsch

Publications and source records attributed to A Rietsch.

7 recordsLinked to original sources

PubMed1999

Importance of redox potential for the in vivo function of the cytoplasmic disulfide reductant thioredoxin from Escherichia coli.

The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.

Cytoplasm↗

PubMed1998

The genetics of disulfide bond metabolism.

Disulfide bonds are required for the stability and function of a large number of proteins. Genetic analysis in combination with biochemical studies have elucidated the main catalysts involved in facilitating these processes in the cell. All enzymes involved in thiol-disulfide metabolism have a conserved active site that consists of two cysteine residues, separated by two intervening amino acids, the Cys-Xaa-Xaa-Cys motif. While these enzymes are capable of catalyzing both disulfide bond formation and reduction, they have evolved to perform one or the other reaction more efficiently. In the cytoplasm, multiple pathways are involved in the reduction of disulfide bonds that occur as part of the catalytic cycle of a variety of metabolic enzymes. In the bacterial periplasm, a system for the efficient introduction as well as isomerization of disulfide bonds is in place. In eukaryotes, disulfide bonds are introduced into proteins in the endoplasmic reticulum. Genetic studies have recently begun to reveal new features of this process. While the enzyme mechanisms of thiol-disulfide oxidoreductases have been the subject of much scrutiny, questions remain regarding where and when they act in vivo, their specificities, and the maintenance of the redox environment that determines their function.

Catalytic Domain↗

PubMed1997

The hydrophilic C-terminal part of the lambda S holin is non-essential for intermolecular interactions.

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the E phi S fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the E phi S fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.

Amino Acid Sequence↗

PubMed1997

Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.

The Escherichia coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase. For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form. Here we present evidence that, in wild-type cells, these two cysteines are reduced. Further, we show that a pathway involving the cytoplasmic proteins thioredoxin reductase and thioredoxin and the cytoplasmic membrane protein DsbD is responsible for the reduction of these cysteines. Thus, reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC. This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway. The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds. Analysis of the activity of mutant forms of DsbC in which either or both of these cysteines have been altered further supports the role of DsbC as a disulfide bond isomerase.

Aprotinin↗

PubMed1996

An in vivo pathway for disulfide bond isomerization in Escherichia coli.

Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

Alkaline Phosphatase↗

PubMed1993

Non-specific hole formation in the Escherichia coli inner membrane by lambda S proteins in independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids.

Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.

Adenosine Triphosphatases↗

PubMed1993

Lambda kil-mediated lysis requires the phage context.

The lambda kil gene has been shown to be responsible for premature lysis effected by addition of chloramphenicol between 15 and 20 min after thermal induction of a lambda prophage. Here, we localized the kil reading frame. The kil gene, represented by lambda orf47, overlaps genes cIII and gam. Expression of the plasmid-borne kil gene resulted in growth arrest, a reduction of colony-forming units and filament formation. However, kil-mediated cell lysis could not be triggered by chloramphenicol when the plasmid borne kil gene was expressed, suggesting that kil-induced cell lysis requires the phage context.

Bacteriophage lambda↗