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Timothy Phillips, Chong Wai Tio, Gregory Omerza, Abhimannyu Rimal, Ravi Kumar Lokareddy, Gino Cingolani, Edward Winter
Smk1 is a MAPK in the yeast Saccharomyces cerevisiae that controls the post-meiotic program of spore formation. Ssp2 is a meiosis-specific protein that activates Smk1 and triggers the autophosphorylation of its activation loop. A fragment of Ssp2 that is sufficient to activate Smk1 contains two segments that resemble RNA Recognition Motifs (RRMs). Mutations in either of these motifs eliminated Ssp2's ability to activate Smk1. In contrast, deletions and insertions within the segment linking the RRM-like motifs only partially reduced the activity of Ssp2...
November 19, 2018: Biochemistry
Luca Dalponte, Anirudra Parajuli, Ellen Younger, Antti Mattila, Jouni Jokela, Matti Wahlsten, Niina Leikoski, Scott A Jarmusch, Kaarina Sivonen, Wael ElSayed Houssen, David P Fewer
Aromatic prenylation is an important step in the biosynthesis of many natural products and leads to an astonishing diversity of chemical structures. Cyanobactin pathways frequently encode aromatic prenyltransferases that catalyze the prenylation of these macrocyclic and linear peptides. Here we characterized the anacyclamide (acy) biosynthetic gene cluster from Anabaena sp. UHCC-0232. Partial reconstitution of the anacyclamide pathway, heterologous expression and in vitro biochemical characterization of the enzyme demonstrate that the AcyF enzyme encoded in this biosynthetic gene cluster is a Trp N-prenyltransferase...
November 19, 2018: Biochemistry
Eugénia Nogueira, Marisa P Sárria, Nuno Gonçalo Azoia, Egipto Ferreira Antunes, Ana Loureiro, Diana Guimarães, Jennifer Noro, Alexandra Rollett, Georg M Guebitz, Artur Cavaco-Paulo
The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. Methotrexate entry into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained doubtful...
November 19, 2018: Biochemistry
Vignesh Kasinath, Simon Poepsel, Eva Nogales
Polycomb group proteins are transcriptional repressors controlling gene expression patterns and maintaining cell type identity. The chemical modifications of histones and DNA by the regulated activity of chromatin modifying enzymes such as Polycomb help establish and maintain such expression patterns. Polycomb repressive complex 2 (PRC2) is the only known methyltransferase specific for histone H3 lysine 27 (H3K27) and catalyzes its tri-methylation leading to the repressive H3K27me3 mark. Structural biology has made important contributions towards understanding the molecular mechanisms that ensure the spatiotemporal regulation of PRC2 activity and the establishment of inactive chromatin domains marked by H3K27me3...
November 19, 2018: Biochemistry
Takehiro Ohta, Tomokazu Shibata, Yasuhiro Kobayashi, Yoshitaka Yoda, Takashi Ogura, Saburo Neya, Akihiro Suzuki, Makoto Seto, Yasuhiko Yamamoto
The molecular mechanism of O2 binding to hemoglobin (Hb) and myoglobin (Mb) is a long-standing issue in the field of bioinorganic and biophysical chemistry. The nature of Fe-O2 bond in oxy Hb and Mb had been extensively investigated by resonance Raman spectroscopy, which assigned the Fe-O2 stretching bands at ∼570 cm-1 . However, resonance Raman assignment of the vibrational mode had been elusive due to the spectroscopic selection rule and to the limited information available about the ground-state molecular structure...
November 19, 2018: Biochemistry
Brinton Seashore-Ludlow, Hanna Axelsson, Helena Almqvist, Björn Dahlgren, Mats Jonsson, Thomas Lundbäck
Evidence of physical interaction with the target protein is essential in the development of chemical probes and drugs. The cellular thermal shift assay (CETSA) allows evaluation of drug binding in live cells but lacks a framework to support quantitative interpretations and comparisons with functional data. We outline an experimental platform for such analysis using human kinase p38α. Systematic variations to the assay's characteristic heat challenge demonstrate an apparent loss of compound potency with an increase in duration or temperature, in line with expectations from the literature for thermal shift assays...
November 19, 2018: Biochemistry
Ying Li, Ke Ke, Robert C Spitale
Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond coding for proteins. Critical to RNA function is its transport to unique subcellular locations, where it can be spatially regulated to perform its biological function. Within this perspective we highlight the techniques developed to image and discover RNAs within their subcellular compartments, and discuss the utility of these techniques in understanding RNA localization.
November 16, 2018: Biochemistry
Sukrut C Kamerkar, Krishnendu Roy, Soumya Bhattacharyya, Thomas J Pucadyil
Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors membrane tube scission, we detect robust GTP- and ATP-dependent as well as nucleotide-independent fission activity in the brain cytosol. Using previously established interacting partner proteins as bait for pulldowns, we attribute the GTP-dependent fission activity to dynamin...
November 16, 2018: Biochemistry
Filippo Fiorentini, Anna-Maria Hatzl, Sandy Schmidt, Simone Savino, Anton Glieder, Andrea Mattevi
CYP153s are bacterial class I P450 enzymes traditionally described as alkane hydroxylases with a high terminal regioselectivity. They have been more recently shown to also catalyze hydroxylations at nonactivated carbon atoms of small heterocycles. The aim of our work was to perform an extensive characterization of this subfamily in order to deliver a toolbox of CYP153 enzymes for further development as biocatalysts. Through the screening of recently sequenced bacterial genomes, 20 CYP153s were selected, comprising 17 single monooxygenase domains and three multidomain variants, where the monooxygenase domain is naturally fused to its redox partners in a single polypeptide chain...
November 16, 2018: Biochemistry
Paul D Carlson, Julius B Lucks
No abstract text is available yet for this article.
November 15, 2018: Biochemistry
Lois Manning, James M Manning
The mild acetylating agent, methyl acetyl phosphate, is used to estimate the pKa values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys epsilon amines have pKa values in the range of 7.8-8.3, which are much lower than currently assumed. When Ser-1 is phosphorylated, the pKa values of these Lys amines are elevated to the range of 8.8-10.3 thus providing the rationale for reports that they are then better substrates for acetyltransferases...
November 15, 2018: Biochemistry
Hanqiao Feng, Bing-Rui Zhou, Yawen Bai
It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh-affinity (equilibrium dissociation constant KD of ~2 x 10-12 M) measured using a single-molecule FÖrster resonance energy transfer (smFRET) method. It was hypothesized that the ultrahigh-affinity and extreme disorder may be required for the chaperone function of ProTα , in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα -H1...
November 15, 2018: Biochemistry
W Andrew Vinyard, Aaron M Fleming, Jingwei Ma, Cynthia J Burrows
Chlamydomonas reinhardtii is a green alga with a very GC-rich genome (67%) and a high density of potential G-quadruplex-forming sequences (PQSs). Using the Ensembl Plants DNA database, 19 PQSs were selected, and their ability to fold in vitro was examined using four experimental methods. Our results support in vitro folding of 18 of the 19 PQSs selected for study. The high physiological polyamine concentrations in C. reinhardtii create unique conditions for studying G4 folding. We investigated whether high polyamine concentrations affect the stability and structural fold of two polymorphic G4s selected from the cohort of PQSs...
November 15, 2018: Biochemistry
Abigail R Green, Cody Freedman, Jennyfer Tena, Benjamin E Tourdot, Benjamin Liu, Michael Holinstat, Theodore R Holman
The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4 . For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0...
November 15, 2018: Biochemistry
Bhanu Pratap Singh Chouhan, Shayida Maimaiti, Madhuri Gade, Paola Laurino
Methyltransferases (MTases) are superfamilies of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM), a nucleoside-based cofactor, to a wide variety of substrates such as DNA, RNA, proteins, small molecules, and lipids. Depending upon their structural features, the MTases can be further classified into different classes; we consider exclusively the largest class of MTases, the Rossmann-fold MTases. It has been shown that the nucleoside cofactor-binding Rossmann enzymes, particularly the nicotinamide adenine dinucleotide (NAD)-, flavin adenine dinucleotide (FAD)-, and SAM-binding MTases enzymes, share common binding motifs that include a Gly-rich loop region that interacts with the cofactor and a highly conserved acidic residue (Asp/Glu) that interacts with the ribose moiety of the cofactor...
November 15, 2018: Biochemistry
Chunhua Liu, Jingwei Weng, Dan Wang, Maohua Yang, Min Jia, Wenning Wang
GoLoco motif-containing proteins regulate the nucleotide-binding state of Gα proteins in various signaling pathways. As guanine nucleotide dissociation inhibitors (GDIs), they bind Gα·GDP and inhibit GDP to GTP exchange. GoLoco proteins show binding selectivity toward different members of the Gα family. Although the Gαi1 ·GDP/RGS14 crystal structure explains the specific binding selectivity of the RGS14 GoLoco domain well, the mechanism of selective binding has not been understood for the more general features of short GoLoco domains found in tandem arrays in proteins like GPSM2/LGN/ dPins and GPSM1/AGS3...
November 15, 2018: Biochemistry
Qingyun Xie, Jing Liu, Yanke Shan, Shouyu Wang, Fei Liu
RNA helicase A (RHA) as a member of the DExH/D-box subgroup of helicase superfamily II is involved in virtually all aspects of RNA metabolism. It exhibits robust RNA helicase activity in vitro. However, little is known about the molecular and physical determinants for RHA substrate recognition and RHA translocation along the nucleic acids. Here, our nondenaturing polyacrylamide gel electrophoresis (PAGE)-based unwinding assays of chemical and structural modified substrates indicate that RHA translocates efficiently along the 3' overhang of RNA, but not DNA, with a requirement of covalent continuity...
November 15, 2018: Biochemistry
Daniel C Bailey, Tabbetha J Bohac, Justin A Shapiro, Daryl E Giblin, Timothy A Wencewicz, Andrew M Gulick
The critical role that iron plays in many biochemical processes has led to an elaborate battle between bacterial pathogens and their hosts to acquire and withhold this critical nutrient. Exploitation of iron nutritional immunity is being increasingly appreciated as a potential antivirulence therapeutic strategy, especially against problematic multidrug resistant Gram-negative pathogens such as Acinetobacter baumannii. To facilitate iron uptake and promote growth, A. baumannii produces a nonribosomally synthesized peptide siderophore called acinetobactin...
November 15, 2018: Biochemistry
Ivan L Volkov, Magnus Johansson
Decades of traditional biochemistry, structural approaches, and, more recently, single-molecule-based in vitro techniques have provided us with an astonishingly detailed understanding of the molecular mechanism of ribosome-catalyzed protein synthesis. However, in order to understand these details in the context of cell physiology and population biology, new techniques to probe the dynamics of molecular processes inside the cell are needed. Recent years' development in super-resolved fluorescence microscopy has revolutionized imaging of intracellular processes, and we now have the possibility to directly peek into the microcosm of biomolecules in their native environment...
November 15, 2018: Biochemistry
Jake N Gonzales, Morgan M Matson, Shota Atsumi
Alarming changes in environmental conditions have prompted significant research into producing renewable commodities from sources other than fossil fuels. One such alternative is CO2 , a determinate greenhouse gas with historically high atmospheric levels. If sequestered, CO2 could be used as a highly renewable feedstock for industrially relevant products and fuels. The vast majority of atmospheric CO2 fixation is accomplished by photosynthetic organisms, which have unfortunately proven difficult to utilize as chassis for industrial production...
November 15, 2018: Biochemistry
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