The gene encoding Desulfovibrio gigas flavoredoxin was deleted to elucidate its physiological role in the sulfate metabolism. Disruption of flr gene strongly inhibited the reduction of thiosulfate and exhibited a reduced growth in the presence of sulfite with lactate as electron donor. The growth with sulfate was not however affected by the lack of this protein. Additionally, flr mutant cells revealed a decrease of about 50% in the H 2 consumption rate using thiosulfate as electron acceptor. Altogether, our results show in vivo that during sulfite respiration, trithionate and thiosulfate are produced and that flavoredoxin is specific for thiosulfate reduction.

Sulfate-reducing bacteria are rich in unique redox proteins and electron carriers that participate in a variety of essential pathways. Several studies have been carried out to characterize these proteins, but the structure and function of many are poorly understood. Many Desulfovibrio species can grow using hydrogen as the sole energy source, indicating that the oxidation of hydrogen with sulfite as the terminal electron acceptor is an energy-conserving mechanism. Flavoredoxin is an FMN-binding protein isolated from the sulfate-reducing bacteria Desulfovibrio gigas that participates in the reduction of bisulfite from hydrogen. Here we report the cloning and sequencing of the flavoredoxin gene. The derived amino acid sequence exhibits similarity to several flavoproteins which are members of a new family of flavin reductases suggested to bind FMN in a novel mode. (C) 2000 Academic Press.

Flavoredoxin participates in Desulfovibrio gigas thiosulfate reduction pathway. Its 3-dimensional model was generated allowing the oxidized riboflavin-5 0 -phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein.Comparison of K d values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were correlated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavoredoxin.

Abstract:Flavoredoxin is an FMN binding protein that functions as an electron carrier in the sulphate metabolism of Desulfovibrio gigas. The neighbouring DNA regions of the gene encoding flavoredoxin were sequenced and characterised. Transcript analysis of the flavoredoxin gene resulted in a positive band corresponding to the size of the coding region, suggesting that flavoredoxin is encoded by a monocystronic unit, as previously suggested by sequence analysis.Analysis of the adjacent DNA regions revealed several interesting genes. The sequenced DNA regions contain nine open reading frames (ORFs) organised in two polycystronic and two monocystronic units. These genes encode proteins involved in different metabolic pathways, namely in DNA methylation, tRNA and rRNA modification, mRNA metabolism, cell division, CoA synthesis and lipoprotein transport across the membrane. Document Type: ArticleLanguage: English

The glomerular mesangial cells (GMC) play a central role in the synthesis and turnover of the glomerular mesangial matrix. The breakdown of the matrix likely depends on the balance between a variety of proteinases including matrix metalloproteinases and their biological inhibitors secreted by the GMC. Therefore, we studied pattern secretion of matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-3, MMP-9 and their biological tissue inhibitor of matrix metalloproteinases (TIMPs), TIMP-1 and TIMP-2 by cultured human GMC. We also measured MMP-l/TIMP-1 complex level in the cell culture supernatants. For this purpose, the GMC were incubated under serum-free conditions with medium (RPMI-1640) alone or in combination with TNF-alpha (30 ng/ml) or PMA (50 ng/ml) for 24,48 and 72 h. The metalloproteinases and their inhibitors were assayed by established ELISA techniques. Our results showed that the lowest and largest secretions were related to MMP-9 and MMP-2, respectively. The results indicated that the MMPs and TIMPs secretion were increased by TNF-alpha (MMP-1, MMP-2, TIMP-1 and TIMP-2) and PMA (MMP-2, TIMP-1 and TIMP-2), significantly (P < 0.05). These results suggest that GMC can synthesis and release various MMPs and their inhibitors (TIMPs) that, in part, control turnover of extracellular matrix proteins.An increased burden of oxidants and depletion of antioxidant defenses may contribute to the age-related decline in adrenal steroidogenesis. I n this study, we sought to determine whether aging influences the sensitivity of the adrenal to LPSstress related activation of NF-kB, a redox-sensitive transcription factor. Young mature (5 mo) and old (24-26 mo) rats were injected without or with LPS and nuclear extracts studied using electrophoretic mobility shift assay (EMSA) andWestern blotting. The adrenals from old animals showed -50% reduction in activation of NF-kB D N A binding activity as determined by EMSA, when treated with LPS as compared to values from the adrenals of young animals. Supershift analysis confirmed the presense of p65 and p50 proteins in D N A complexes. Aging decreased both total and nuclear levels of p65, but had no effect o n IkB inhibitory proteins. These findings suggest that aging-induced increases in oxidative stress might be related to alterations in NF-kB signaling, which could contribute to the adverse affects of aging on adrenal steroidogenesis. Desulfovibrio gigus is a sulphate reducing bacterium, very rich in several proteins involved in oxido-reduction reactions. These proteins show a great variety of prosthetic groups, playing an important role in the biochemical understanding of this bacterium. Some of these proteins are very well characterized, however, the function of many remains unclear. The recombinant overexpressed proteins help us to get information on its function and structure. One of these important proteins is the flavoredoxin. Flavoredoxin is a FMN binding protein that participates in the reduction of bisulphite from molecular hydrogen. According to the significant homolo...