Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity.

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.

Comparative structural analysis of oxidized and reduced thioredoxin from Drosophila melanogaster.

Thioredoxins (Trx) participate in essential antioxidant and redox-regulatory processes via a pair of conserved cysteine residues. In dipteran insects like Drosophila and Anopheles, which lack a genuine glutathione reductase (GR), thioredoxins fuel the glutathione system with reducing equivalents. Thus, characterizing Trxs from these organisms contributes to our understanding of redox control in GR-free systems and provides information on novel targets for insect control. Cytosolic Trx of Drosophila melanogaster (DmTrx) is the first thioredoxin that was crystallized for X-ray diffraction analysis in the reduced and in the oxidized form. Comparison of the resulting structures shows rearrangements in the active-site regions. Formation of the C32-C35 disulfide bridge leads to a rotation of the side-chain of C32 away from C35 in the reduced form. This is similar to the situation in human Trx and Trx m from spinach chloroplasts but differs from Escherichia coli Trx, where it is C35 that moves upon change of the redox state. In all four crystal forms that were analysed, DmTrx molecules are engaged in a non-covalent dimer interaction. However, as demonstrated by gel-filtration analyses, DmTrx does not dimerize under quasi in vivo conditions and there is no redox control of a putative monomer/dimer equilibrium. The dimer dissociation constants K(d) were found to be 2.2mM for reduced DmTrx and above 10mM for oxidized DmTrx as well as for the protein in the presence of reduced glutathione. In human Trx, oxidative dimerization has been demonstrated in vitro. Therefore, this finding may indicate a difference in redox control of GR-free and GR-containing organisms.

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge.

This communication presents a short outline of the current knowledge on the molecular basis of P(II) signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P(II) signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P(II) signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P(II) phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P(II) (P(II)-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P(II)-P dephosphorylation, novel insights into the signals, to which P(II)-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P(II)-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P(II)-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P(II)-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.

The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII.

The family of PII signal transduction proteins consists of one of the most highly conserved signalling proteins in nature. The cyanobacterial PII homologue transmits signals on the nitrogen and carbon status of the cells through phosphorylation of a seryl residue. Recently, we identified a protein phosphatase 2C (PP2C) homologue from the cyanobacterium Synechocystis PCC 6803, termed PphA, to be the cellular phospho-PII (PII-P) phosphatase. In this investigation, we characterized the enzymatic properties of PphA and investigated the regulation of its catalytic activity towards PII-P. PphA dephosphorylates phosphocasein and PII-P with similar efficiency in a strictly Mg2+- or Mn2+-dependent reaction. Low-molecular-weight phosphorylated molecules are poor substrates for PphA. Its reactivity towards PII-P, but not towards phosphocasein, is inhibited by various nucleotides, suggesting that this effect is based on specific properties of the PII protein. The inhibitory effect of ATP can be strongly enhanced by the addition of 2-oxoglutarate or oxaloacetate. At low concentrations of 2-oxoglutarate, changes in the ATP levels within the physiological range affect the degree of PII-Pase inhibition, whereas at 2-oxoglutarate levels beyond 0.1 mM, inhibition is almost complete at very low ATP levels. This suggests that PII dephosphorylation is not only sensitive to 2-oxoglutarate and oxaloacetate levels, it also integrates signals from the energy charge of the cells under specific cellular conditions.