Purification, crystallization and preliminary X-ray analysis of the DndE protein from Salmonella enterica serovar Cerro 87, which is involved in DNA phosphorothioation

State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.53). 11/2011; 67(Pt 11):1440-2. DOI: 10.1107/S1744309111036694
Source: PubMed


The phenomenon of DNA phosphorothioation (DNA sulfur modification) is widespread among prokaryotes and may serve as a mechanism to restrict gene transfer among bacteria. DndE is one of five essential proteins that are required for the DNA phosphorothioation process. However, its exact biochemical role in sulfur modification of DNA remains unclear. In this study, the DndE protein homologue from Salmonella enterica serovar Cerro 87 was overexpressed, purified and crystallized. The crystals of the DndE protein diffracted to 2.7 Å resolution and belonged to space group P3(1)21. These results will facilitate detailed structural analysis of DndE and further elucidation of its biochemical function.

5 Reads
  • Source
    • "Up to now, there has been not much structural investigation on proteins involved in DNA phosphorothioation [19]. In this study, we determined the crystal structure of DndA from Streptomyces lividans, to a resolution of 2.4 Å. "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA.
    PLoS ONE 05/2012; 7(5):e36635. DOI:10.1371/journal.pone.0036635 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphorothioate modification of DNA and the corresponding DNA degradation (Dnd) phenotype that occurs during gel electrophoresis are caused by dnd genes. Although widely distributed among Bacteria and Archea, dnd genes have been found in only very few, taxonomically unrelated, bacterial species so far. Here, we report the presence of dnd genes and their associated Dnd phenotype in two Flavobacterium species. Comparison with dnd gene clusters previously described led us to report a non canonical genetic organization and to identify a gene likely encoding an hybrid DndE protein. Hence, we showed that dnd genes are also present in members of the family Flavobacteriaceae, a bacterial group occurring in a variety of habitats with an interesting diversity of lifestyle. Two main types of genomic organization of dnd loci were uncovered probably denoting their spreading in the phylum Bacteroidetes via distinct genetic transfer events. This article is protected by copyright. All rights reserved.
    FEMS Microbiology Letters 08/2013; 348(1). DOI:10.1111/1574-6968.12239 · 2.12 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Prokaryotes protect their genomes from foreign DNA with a diversity of defense mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT-modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of >600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, over-expression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification.
    Molecular Microbiology 07/2014; 93(4). DOI:10.1111/mmi.12692 · 4.42 Impact Factor