The barley grain thioredoxin system - an update.

Thioredoxin reduces disulfide bonds and play numerous important functions in plants. In cereal seeds, cytosolic h-type thioredoxin facilitates the release of energy reserves during the germination process and is recycled by NADPH-dependent thioredoxin reductase. This review presents a summary of the research conducted during the last ten years to elucidate the structure and function of the barley seed thioredoxin system at the molecular level combined with proteomic approaches to identify target proteins

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from <em>E. coli</em>

<div><p>YjdL from <em>E. coli</em> is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical <em>E. coli</em> POT, were compared in light of the crystal structure of a POT from <em>Shewanella oneidensis</em>. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.</p> </div

functional analyses of YdgR variants.

<p>(A) Representative western blots of YdgR mutants. (B) β-Ala-Lys(AMCA) uptake (0.2 mM, 5 min) by YdgR mutants in uptake buffer, pH 6.5. Error bars indicate SEM (n ≥3). Lys133Arg and Lys133Gln are not significantly different, P>0.05, from background levels (pTTQ18).</p

Primers used for site-directed mutagenesis.

<p>IC₅₀ values (mM) with SEM (n ≥3) of selected dipeptides tested on YjdL and YdgR variants.</p>a<p>IC₅₀ values determined at 0.2 mM β-Ala-Lys(AMCA) and 5 min incubation.</p>b<p>IC₅₀ values determined at 0.5 mM β-Ala-Lys(AMCA) and 15 min incubation.</p