Purification and crystallization of<i>Bacillus subtilis</i>NrnA, a novel enzyme involved in nanoRNA degradation

Nelersa, Claudiu M.; Schmier, Brad J.; Malhotra, Arun

doi:10.1107/s1744309111026509

Cited by 4 publications

(8 citation statements)

References 15 publications

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“…Bacillus NrnA was crystallized in the P 2 1 space group with four molecules per asymmetric unit 27 . The 2.0 Å structure of native apo-NrnA was solved using phases obtained by molecular replacement with the structure of a homologous unpublished S. haemolyticus protein (PDB ID: 3DEV).…”

Section: Resultsmentioning

confidence: 99%

“…The NrnA active site is formed by four aspartate residues (D24, D26, D80 and D156) and three histidines (H103, H104 and H20) that combine to generate a shallow cleft in the DHH domain. To investigate metal binding at the NrnA active site, NrnA H103A (purified in the presence of 2 mM EDTA) was crystallized as described and soaked with 10 mM MnCl 2 and 10 mM pAp 27 . Strong 2 Fo - Fc electron density (ranging from 9.0 σ to 6.5 σ ) was seen at two positions in each active site for all four molecules in the asymmetric unit.…”

Section: Resultsmentioning

confidence: 99%

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Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA

Schmier

Nelersa

Malhotra

2017

Sci Rep

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NanoRNAs are RNA fragments 2 to 5 nucleotides in length that are generated as byproducts of RNA degradation and abortive transcription initiation. Cells have specialized enzymes to degrade nanoRNAs, such as the DHH phosphoesterase family member NanoRNase A (NrnA). This enzyme was originally identified as a 3′ → 5′ exonuclease, but we show here that NrnA is bidirectional, degrading 2–5 nucleotide long RNA oligomers from the 3′ end, and longer RNA substrates from the 5′ end. The crystal structure of Bacillus subtilis NrnA reveals a dynamic bi-lobal architecture, with the catalytic N-terminal DHH domain linked to the substrate binding C-terminal DHHA1 domain via an extended linker. Whereas this arrangement is similar to the structure of RecJ, a 5′ → 3′ DHH family DNase and other DHH family nanoRNases, Bacillus NrnA has gained an extended substrate-binding patch that we posit is responsible for its 3′ → 5′ activity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA

Schmier

Nelersa

Malhotra

2017

Sci Rep

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“…The presence of selenite in the culture medium of B. subtilis causes structural changes in cells, increase in the content of thioredoxin and the NADP-thioredoxine reductase enzyme associated with it (Garbisu et al, 1999;Nelersa et al, 2011). Selenites can contribute to the oxidation of thiols to sulfenic acid.…”

Section: Discussionmentioning

confidence: 99%

“…Thioredoxin can reduce protein disulfides through redox-active dithiols. In B. subtilis, thioredoxin is induced in the presence of selenite (Garbisu et al, 1999;Nelersa et al, 2011). B. subtilis is a subgroup of microorganisms which has the property to metabolise selenium.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of effects of selenium nanoparticles on Bacillus subtilis

Tymoshok

Kharchuk

Kaplunenko

et al. 2019

Regul. Mech. Biosyst.

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The present study was performed to characterize of selenium nanoparticles (Nano-Se) which were synthesized by pulsed laser ablation in liquids to obtain the aqueous selenium citrate solution. The study was conducted using bacteriological and electronic-microscopic methods. Transmission electron microscopy (TEM) and spectroscopy analyses demonstrated that nano-selenium particles obtained by the method of selenium ablation had the size of 4–8 nm. UV-Visible Spectrum colloidal solution Nano-Se exhibited absorption maxima at 210 nm. To clarify some effects of the action of Nano-Se on Bacillus subtilis, we investigated the interaction of Nano-Se with B. subtilis IMV B-7392 before and after incubation with Nano-Se, examining TEM images. It has been shown that exposure to B. subtilis IMV B-7392 in the presence of Nano-Se is accompanied by the rapid uptake of Nano-Se by bacterial culture. TEM analysis found that the electron-dense Nano-Se particles were located in the intracellular spaces of B. subtilis IMV B-7392. That does not lead to changes in cultural and morphological characteristics of B. subtilis IMV B-7392. Using TEM, it has been shown that penetration of nanoparticles in the internal compartments is accompanied with transient porosity of the cell membrane of B. subtilis IMV B-7392 without rupturing it. The effective concentration of Nano-Se 0.2 × 10–3 mg/mL was found to increase the yield of biologically active substances of B. subtilis. In order to create probiotic nano-selenium containing products, the nutrient medium of B. subtilis IMV B-7392 was enriched with Nano-Se at 0.2 × 10–3 mg/mL. It was found that particles Nano-Se are non-toxic to the culture and did not exhibit bactericidal or bacteriostatic effects. The experimentally demonstrated ability of B. subtilis to absorb selenium nanoparticles has opened up the possibility of using Nano-Se as suitable drug carriers.

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“…Moreover, PnpA has also been implicated in DNA repair (70). Finally, we decided to include the nano-RNase NrnA, which degrades and thus recycles bi-and oligoribonucleotides (71). This enzyme is conserved in all groups of mollicutes despite their significant genome reduction (42).…”

Section: Mrna Folding and Degradationmentioning

confidence: 99%

The Blueprint of a Minimal Cell: MiniBacillus

Reuß

Commichau

Gundlach

et al. 2016

Microbiol Mol Biol Rev

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SUMMARYBacillus subtilisis one of the best-studied organisms. Due to the broad knowledge and annotation and the well-developed genetic system, this bacterium is an excellent starting point for genome minimization with the aim of constructing a minimal cell. We have analyzed the genome ofB. subtilisand selected all genes that are required to allow life in complex medium at 37°C. This selection is based on the known information on essential genes and functions as well as on gene and protein expression data and gene conservation. The list presented here includes 523 and 119 genes coding for proteins and RNAs, respectively. These proteins and RNAs are required for the basic functions of life in information processing (replication and chromosome maintenance, transcription, translation, protein folding, and secretion), metabolism, cell division, and the integrity of the minimal cell. The completeness of the selected metabolic pathways, reactions, and enzymes was verified by the development of a model of metabolism of the minimal cell. A comparison of theMiniBacillusgenome to the recently reported designed minimal genome ofMycoplasma mycoidesJCVI-syn3.0 indicates excellent agreement in the information-processing pathways, whereas each species has a metabolism that reflects specific evolution and adaptation. The blueprint ofMiniBacilluspresented here serves as the starting point for a successive reduction of theB. subtilisgenome.

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Purification and crystallization ofBacillus subtilisNrnA, a novel enzyme involved in nanoRNA degradation

Cited by 4 publications

References 15 publications

Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA

Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA

Evaluation of effects of selenium nanoparticles on Bacillus subtilis

The Blueprint of a Minimal Cell: MiniBacillus

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