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 Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

2019 ◽  
Author(s):  
Sindhu Carmen Sivakumaren ◽  
Hyeseok Shim ◽  
Tinghu Zhang ◽  
Fleur M. Ferguson ◽  
Mark R. Lundquist ◽  
...  
Keyword(s):  
Cancer Metabolism ◽  
Target Genes ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Cancer Cell Proliferation ◽  
Lipid Kinase ◽  
Covalent Inhibitors ◽  
Lipid Kinases ◽  
Disordered Loop

SummaryThe PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K’s reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

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.

A Pyridinic Fe-N4 Macrocycle Effectively Models the Active Sites in Fe/N-Doped Carbon Electrocatalysts

2020 ◽  
Author(s):  
Travis Marshall-Roth ◽  
Nicole J. Libretto ◽  
Alexandra T. Wrobel ◽  
Kevin Anderton ◽  
Nathan D. Ricke ◽  
...  
Keyword(s):  
Oxygen Reduction ◽  
Active Sites ◽  
Catalytic Properties ◽  
Reduction Reaction ◽  
Iron Phthalocyanine ◽  
Electrochemical Studies ◽  
Onset Potential ◽  
Nitrogen Doped Carbon ◽  
Iron Active Sites

Iron- and nitrogen-doped carbon (Fe-N-C) materials are leading candidates to replace platinum in fuel cells, but their active site structures are poorly understood. A leading postulate is that iron active sites in this class of materials exist in an Fe-N<sub>4</sub> pyridinic ligation environment. Yet, molecular Fe-based catalysts for the oxygen reduction reaction (ORR) generally feature pyrrolic coordination and pyridinic Fe-N<sub>4</sub> catalysts are, to the best of our knowledge, non-existent. We report the synthesis and characterization of a molecular pyridinic hexaazacyclophane macrocycle, (phen<sub>2</sub>N<sub>2</sub>)Fe, and compare its spectroscopic, electrochemical, and catalytic properties for oxygen reduction to a prototypical Fe-N-C material, as well as iron phthalocyanine, (Pc)Fe, and iron octaethylporphyrin, (OEP)Fe, prototypical pyrrolic iron macrocycles. N 1s XPS signatures for coordinated N atoms in (phen<sub>2</sub>N<sub>2</sub>)Fe are positively shifted relative to (Pc)Fe and (OEP)Fe, and overlay with those of Fe-N-C. Likewise, spectroscopic XAS signatures of (phen<sub>2</sub>N<sub>2</sub>)Fe are distinct from those of both (Pc)Fe and (OEP)Fe, and are remarkably similar to those of Fe-N-C with compressed Fe–N bond lengths of 1.97 Å in (phen<sub>2</sub>N<sub>2</sub>)Fe that are close to the average 1.94 Å length in Fe-N-C. Electrochemical studies establish that both (Pc)Fe and (phen<sub>2</sub>N<sub>2</sub>)Fe have relatively high Fe(III/II) potentials at ~0.6 V, ~300 mV positive of (OEP)Fe. The ORR onset potential is found to directly correlate with the Fe(III/II) potential leading to a ~300 mV positive shift in the onset of ORR for (Pc)Fe and (phen<sub>2</sub>N<sub>2</sub>)Fe relative to (OEP)Fe. Consequently, the ORR onset for (phen<sub>2</sub>N<sub>2</sub>)Fe and (Pc)Fe is within 150 mV of Fe-N-C. Unlike (OEP)Fe and (Pc)Fe, (phen<sub>2</sub>N<sub>2</sub>)Fe displays excellent selectivity for 4-electron ORR with <4% maximum H<sub>2</sub>O<sub>2</sub> production, comparable to Fe-N-C materials. The aggregate spectroscopic and electrochemical data establish (phen<sub>2</sub>N<sub>2</sub>)Fe as a pyridinic iron macrocycle that effectively models Fe-N-C active sites, thereby providing a rich molecular platform for understanding this important class of catalytic materials.<p><b></b></p>

Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 541-551 ◽  
Author(s):  
N. E. COLLINS ◽  
B. A. ALLSOPP
Keyword(s):  
Genetic Recombination ◽  
Large Subunit ◽  
Small Subunit ◽  
Internal Transcribed Spacers ◽  
Rrna Genes ◽  
Gene Pools ◽  
Theileria Parva ◽  
Causative Agents ◽  
Internal Transcribed Spacer Sequences

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5·8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93·5% of T. p. parva isolates examined were detected by probe TPP1 and 81·8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

Defect engineering and characterization of active sites for efficient electrocatalysis

Nanoscale ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 3327-3345
Author(s):  
Xuecheng Yan ◽  
Linzhou Zhuang ◽  
Zhonghua Zhu ◽  
Xiangdong Yao
Keyword(s):  
Transition Metal ◽  
Active Sites ◽  
Defect Engineering ◽  
Metal Free

This review highlights recent advancements in defect engineering and characterization of both metal-free carbons and transition metal-based electrocatalysts.

Structural Characterization of Complex Bacterial Glycolipids by Fourier Transform Mass Spectrometry

2005 ◽  
Vol 11 (5) ◽  
pp. 535-546 ◽  
Author(s):  
Anna Kondakov ◽  
Buko Lindner
Keyword(s):  
Mass Spectrometry ◽  
Fourier Transform ◽  
Structural Information ◽  
Adaptive Immune System ◽  
Ion Cyclotron Resonance ◽  
Bacterial Membranes ◽  
Multiphoton Dissociation ◽  
The One ◽  
And Function

Bacterial glycolipids are complex amphiphilic molecules which are, on the one hand, of utmost importance for the organization and function of bacterial membranes and which, on the other hand, play a major role in the activation of cells of the innate and adaptive immune system of the host. Already small alterations to their chemical structure may influence the biological activity tremendously. Due to their intrinsic biological heterogeneity [number and type of fatty acids, saccharide structures and substitution with for example, phosphate ( P), 2-aminoethyl-(pyro)phosphate groups ( P-Etn) or 4-amino-4-deoxyarabinose (Ara4N)], separation of the different components are a prerequisite for unequivocal chemical and nuclear magnetic resonance structural analyses. In this contribution, the structural information which can be obtained from heterogenous samples of glycolipids by Fourier transform (FT) ion cyclotron resonance mass spectrometric methods is described. By means of recently analysed complex biological samples, the possibilities of high-resolution electrospray ionization FT-MS are demonstrated. Capillary skimmer dissociation, as well as tandem mass spectrometry (MS/MS) analysis utilizing collision-induced dissociation and infrared multiphoton dissociation, are compared and their advantages in providing structural information of diagnostic importance are discussed.

Diffuse Reflectance FT-IR Characterization of Active Sites under Reaction Conditions: The Production of Oxygenates in the CO/H2 Reaction

1994 ◽  
Vol 48 (10) ◽  
pp. 1208-1212 ◽  
Author(s):  
J. J. Benítez ◽  
I. Carrizosa ◽  
J. A. Odriozola
Keyword(s):  
Infrared Spectra ◽  
Active Sites ◽  
Diffuse Reflectance ◽  
Reaction Conditions ◽  
In Situ Drifts ◽  
Ft Ir

The reactivity of a Lu2O3-promoted Rh/Al2O3 catalyst in the CO/H2 reaction is reported. Methane, heavier hydrocarbons, methanol, and ethanol are obtained. In situ DRIFTS has been employed to record the infrared spectra under the actual reaction conditions. The structure of the observed COads DRIFTS bands has been resolved into its components. The production of oxygenates (methanol and ethanol) has been correlated with the results of the deconvolution calculation. Specific sites for the production of methanol and ethanol in the CO/H2 reaction over a Rh,Lu2O3/Al2O3 catalyst are proposed.

Identification and characterization of two novel calpain large subunit genes

Gene ◽  
2001 ◽  
Vol 274 (1-2) ◽  
pp. 245-252 ◽  
Author(s):  
T.Neil Dear ◽  
Thomas Boehm
Keyword(s):  
Large Subunit ◽  
Identification And Characterization

A High-Throughput Liposome Substrate Assay with Automated Lipid Extraction Process for PI 3-Kinase

2008 ◽  
Vol 13 (9) ◽  
pp. 906-911 ◽  
Author(s):  
Trupti Lingaraj ◽  
John Donovan ◽  
Zhi Li ◽  
Ping Li ◽  
Amanda Doucette ◽  
...  
Keyword(s):  
High Throughput ◽  
Lipid Extraction ◽  
Extraction Process ◽  
Enzyme Mechanism ◽  
Class I ◽  
Lipid Kinase ◽  
Chromatography Analysis ◽  
Regulate Cell Growth ◽  
Lipid Kinases ◽  
Layer Chromatography

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P2 to PI (3,4,5)P3, which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110α, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases. ( Journal of Biomolecular Screening 2008:906-911)

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