scholarly journals Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage

2019 ◽  
Vol 47 (17) ◽  
pp. 9448-9463 ◽  
Author(s):  
Carl J Schiltz ◽  
April Lee ◽  
Edward A Partlow ◽  
Christopher J Hosford ◽  
Joshua S Chappie
Keyword(s):  
Spatial Organization ◽  
Structural Information ◽  
Domain Architecture ◽  
Underlying Mechanism ◽  
Nuclease Activity ◽  
Catalytic Core ◽  
Metal Catalysis ◽  
Catalysis Mechanism

Abstract Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.

scholarly journals Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency

2017 ◽  
Vol 114 (18) ◽  
pp. 4739-4744 ◽  
Author(s):  
Megan Mayerle ◽  
Madhura Raghavan ◽  
Sarah Ledoux ◽  
Argenta Price ◽  
Nicholas Stepankiw ◽  
...  
Keyword(s):  
High Efficiency ◽  
Catalytic Efficiency ◽  
Mrna Splicing ◽  
Catalytic Core ◽  
Eukaryotic Gene ◽  
Functional Classes ◽  
Reporter Assays

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the “master regulator” of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome’s catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome “toggles” between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

scholarly journals Purification and Characterization of aBacillus subtilis168 Nuclease, YokF, Involved in Chromosomal DNA Degradation and Cell Death Caused by Thermal Shock Treatments

2001 ◽  
Vol 276 (50) ◽  
pp. 47046-47051 ◽  
Author(s):  
Jin J. Sakamoto ◽  
Miho Sasaki ◽  
Tetsuaki Tsuchido
Keyword(s):  
Thermal Shock ◽  
Nuclease Activity ◽  
Dna Degradation ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Purification And Characterization ◽  
Membrane Perturbation

We purified and characterized a 39-kDaBacillus subtilis168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatmentsin vivo. The nuclease activity was inhibitedin vitroby aurintricalboxylic acid but not by Zn2+. By the mutant analysis, we identified the 39-kDa nuclease as a product ofyokFgene. TheyokFgene contained a putative lipoprotein signal peptide motif. Afterin vivoexposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in theyokFmutant, which demonstrated about a 2–10-fold higher survival rate than the wild type. TheyokFmutant was found to be more sensitive to mitomycin C than the wild type. The transformation efficiency of theyokFmutant was about 10 times higher than that of the wild type. It is suggested that whenB. subtiliscells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.

scholarly journals Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Martin Alcorlo ◽  
Daniel Straume ◽  
Joe Lutkenhaus ◽  
Leiv Sigve Håvarstein ◽  
Juan A. Hermoso
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Structural Characterization ◽  
Bound States ◽  
Structural Information ◽  
Structural Plasticity ◽  
Membrane Complex

ABSTRACT FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology. IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo. Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.

scholarly journals Structural characterization of the essential cell division protein FtsE and its interaction with FtsX in Streptococcus pneumoniae

2020 ◽  
Author(s):  
Martin Alcorlo ◽  
Daniel Straume ◽  
Joe Lutkenhaus ◽  
Leiv Sigve Håvarstein ◽  
Juan A. Hermoso
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Structural Characterization ◽  
Structural Information ◽  
Structural Plasticity ◽  
Central Process ◽  
Membrane Complex

ABSTRACTFtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily, where FtsX is an integral membrane protein and FtsE is an ATPase, required for mechanotransmission of the signal from the cytosol through the membrane, to regulate the activity of cell-wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium, Streptococcus pneumoniae and interact with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report the high-resolution structures of pneumococcal FtsE in complex with different nucleotides. Structural analysis reveals that FtsE contains all the conserved structural motifs associated with ATPase activity, and allowed interpretation of the in vivo dimeric arrangement in both ADP and ATP states. Interestingly, three specific FtsE regions were identified with high structural plasticity that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for FtsE contact, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCEBacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, play a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the Type VII family of the ABC-superfamily but instead transporter, couple ATP hydrolysis by FtsE to mechanically transduce a conformational signal to activate PG hydrolases. So far, no structural information is available for FtsE. Here we provide the structural characterization of FtsE confirming its ATPase nature and revealing regions with high structural plasticity key for FtsX binding. The complementary region in FtsX has been also identified and validated in vivo. Our results provide evidences on how difference between ATP and ADP states in FtsE would dramatically alter FtsEX interaction with PG hydrolase PcsB in pneumococcal division.

Antidepressant-like effect of Cecropia glazioui Sneth and its constituents – In vivo and in vitro characterization of the underlying mechanism

Phytomedicine ◽  
2007 ◽  
Vol 14 (6) ◽  
pp. 396-402 ◽  
Author(s):  
F.F. Rocha ◽  
M.T.R. Lima-Landman ◽  
C. Souccar ◽  
M.M. Tanae ◽  
T.C.M. De Lima ◽  
...  
Keyword(s):  
Underlying Mechanism ◽  
In Vitro Characterization

scholarly journals The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Shi-Hui Dong ◽  
Weixin Tang ◽  
Tiit Lukk ◽  
Yi Yu ◽  
Satish K Nair ◽  
...  
Keyword(s):  
Active Sites ◽  
Structural Information ◽  
Large Subunit ◽  
Mammalian Target Of Rapamycin ◽  
Cyclization Reactions ◽  
Catalytic Core ◽  
Lipid Kinase ◽  
Modified Peptides ◽  
Lipid Kinases

The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

scholarly journals Structural library and visualization of endogenously oxidized phosphatidylcholines using mass spectrometry-based techniques

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuta Matsuoka ◽  
Masatomo Takahashi ◽  
Yuki Sugiura ◽  
Yoshihiro Izumi ◽  
Kazuhiro Nishiyama ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Peroxidation ◽  
Liver Injury ◽  
Analytical Methods ◽  
High Resolution Mass Spectrometry ◽  
Structural Information ◽  
Ms Imaging ◽  
Specific Lipid

AbstractAlthough oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Comparison of Immunological and Functional Assays for Measurement of Soluble Fibrin

1995 ◽  
Vol 74 (02) ◽  
pp. 673-679 ◽  
Author(s):  
C E Dempfle ◽  
S A Pfitzner ◽  
M Dollman ◽  
K Huck ◽  
G Stehle ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Low Grade ◽  
Plasma Samples ◽  
Normal Range ◽  
Soluble Fibrin ◽  
Discriminating Power ◽  
Fibrin Monomer ◽  
Cerebral Ischemic

SummaryVarious assays have been developed for quantitation of soluble fibrin or fibrin monomer in clinical plasma samples, since this parameter directly reflects in vivo thrombin action on fibrinogen. Using plasma samples from healthy blood donors, patients with cerebral ischemic insult, patients with septicemia, and patients with venous thrombosis, we compared two immunologic tests using monoclonal antibodies against fibrin-specific neo-epitopes, and two functional tests based on the cofactor activity of soluble fibrin complexes in tPA-induced plasminogen activation. Test A (Enzymun®-Test FM) showed the best discriminating power among normal range and pathological samples. Test B (Fibrinostika® soluble fibrin) clearly separated normal range from pathological samples, but failed to discriminate among samples from patients with low grade coagulation activation in septicemia, and massive activation in venous thrombosis. Functional test C (Fibrin monomer test Behring) displayed good discriminating power between normal and pathological range samples, and correlated with test A (r = 0.61), whereas assay D (Coa-Set® Fibrin monomer) showed little discriminating power at values below 10 μg/ml and little correlation with other assays. Standardization of assays will require further characterization of analytes detected.

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