A thioredoxin with antioxidant activity identified from Eriocheir sinensis

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 51 untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepato-pancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge. (C) 2009 Elsevier Ltd. All rights reserved.

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

Diversity and Distribution of Sediment NirS-Encoding Bacterial Assemblages in Response to Environmental Gradients in the Eutrophied Jiaozhou Bay, China

A gene-clone-library-based molecular approach was used to study the nirS-encoding bacteria-environment relationship in the sediments of the eutrophic Jiaozhou Bay. Diverse nirS sequences were recovered and most of them were related to the marine cluster I group, ubiquitous in estuarine, coastal, and marine environments. Some NirS sequences were unique to the Jiaozhou Bay, such as the marine subcluster VIIg sequences. Most of the Jiaozhou Bay NirS sequences had their closest matches originally detected in estuarine and marine sediments, especially from the Chesapeake Bay, indicating similarity of the denitrifying bacterial communities in similar coastal environments in spite of geographical distance. Multivariate statistical analyses indicated that the spatial distribution of the nirS-encoding bacterial assemblages is highly correlated with environmental factors, such as sediment silt content, NH4+ concentration, and OrgC/OrgN. The nirS-encoding bacterial assemblages in the most hypernutrified stations could be easily distinguished from that of the least eutrophic station. For the first time, the sedimentological condition was found to influence the structure and distribution of the sediment denitrifying bacterial community.