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Huan Huang, Avery Lee McIntosh, Gregory Gerald Martin, Lawrence Dangott, Ann B Kier, Friedhelm Schroeder
Although serum Δ9-tetrahydrocannabinol (Δ9-THC) undergoes rapid hepatic clearance and metabolism, almost nothing is known regarding the mechanism(s) whereby this highly lipophilic phytocannabinoid is transported for metabolism/excretion. A novel NBD-arachidonoylethanolamide (NBD-AEA) fluorescence displacement assay showed that liver fatty acid binding protein (FABP1), the major hepatic endocannabinoid (EC) binding protein, binds the first major metabolite of Δ9-THC (Δ9-THC-OH) as well as Δ9-THC itself...
September 20, 2018: Biochemistry
Ting Yao, Yiyou Huang, Meng Zhang, Yujuan Chen, Hairun Pei, Jianyou Shi, Huanchen Wang, Yousheng Wang, Hengming Ke
The cAMP signaling system plays important roles in the physiological processes of pathogen yeast Candida albicans, but its functional mechanism has not been well illustrated. Here, we report the enzymatic characterization and crystal structures of Candida albicans phosphodiesterase 2 (caPDE2) in the unliganded and IBMX complexed forms. CaPDE2 is a monomer in liquid and crystal states and specifically hydrolyzes cAMP with KM of 35 nM. It does not effectively hydrolyze cGMP as shown by 1.32x105 fold specificity of cAMP/cGMP...
September 19, 2018: Biochemistry
Krishna Mohan Sepuru, Krishna Rajarathnam
Hydrogen-bonding and ionic interactions play fundamental roles in macromolecular recognition and function. In contrast to lysines and arginines, how histidines mediate these interactions is less well understood due to the unique properties of its side chain imidazole that include an aromatic ring with two titratable nitrogens, a pKa that can vary significantly, and the ability to exist in three distinct forms: protonated imidazolium and two tautomeric neutral (Nδ1 and Nε2) states. Here, we characterized the structural features of histidines in the chemokines CXCL8 and CXCL1 in the free, GAG heparin-bound, and CXCR2 receptor N-terminal domain-bound states using solution NMR spectroscopy...
September 19, 2018: Biochemistry
Paul Santner, Joao Miguel da Silva Martins, Jonas Striegler Laursen, Lars Behrendt, Leise Riber, Christian A Olsen, Isaiah T Arkin, Jakob Rahr Winther, Martin Willemoës, Kresten Lindorff-Larsen
The M2 protein is an important target for drugs in the fight against the influenza virus. Due to the emergence of resistance against antivirals directed towards the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays to test potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In the present work, we describe a fluorescence-sensor-based assay, which we termed 'pHlux', that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli...
September 19, 2018: Biochemistry
Christopher L Seiler, Jenna Fernandez, Zoe Koerperich, Molly P Andersen, Delshanee Kotandeniya, Megin E Nguyen, Yuk Yin Sham, Natalia Y Tretyakova
A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten Eleven Translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation...
September 19, 2018: Biochemistry
Paul Santner, Joao Miguel da Silva Martins, Caroline Kampmeyer, Rasmus Hartmann-Petersen, Jonas Striegler Laursen, Amelie Stein, Christian A Olsen, Isaiah T Arkin, Jakob Rahr Winther, Martin Willemoës, Kresten Lindorff-Larsen
The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life-span of a drug in influenza treatment. New M2 inhibitors have been reported that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli based, to enable the study of M2 function and inhibition...
September 19, 2018: Biochemistry
Edwin R Ragwan, Eri Arai, Yan Kung
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the first committed step of the mevalonate pathway, which is used across biology in the biosynthesis of countless metabolites. HMGR consumes 2 equiv of the cofactor NAD(P)H to perform the four-electron reduction of HMG-CoA to mevalonate toward the production of steroids and isoprenoids, the largest class of natural products. Recent structural data have shown that HMGR contains a highly mobile C-terminal domain (CTD) that is believed to adopt many different conformations to permit binding and dissociation of the substrate, cofactors, and products at specific points during the reaction cycle...
September 19, 2018: Biochemistry
Adrian G Torres, Thomas F Wulff, Marta Rodríguez-Escribà, Noelia Camacho, Lluís Ribas de Pouplana
Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNAArg ACG and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction...
September 19, 2018: Biochemistry
Brian W Graham, Michael E Bougoulias, Katie L Dodge, Carly T Thaxton, Danae Olaso, Yeqing Tao, Nicolas L Young, Alan G Marshall, Michael A Trakselis
A growing body of evidence supports a steric exclusion and wrapping model for DNA unwinding in which hexameric helicases interact with the excluded single-stranded DNA (ssDNA) in addition to the encircled strand. Interactions with the excluded ssDNA have been shown to be mediated primarily by electrostatic interactions, but base stacking with surface-exposed tyrosine residues is an alternative hypothesis. Here, we mutated several external tyrosine and positively charged residues from full-length Sulfolobus solfataricus MCM along the proposed path of excluded strand binding and assessed their impact on DNA unwinding...
September 19, 2018: Biochemistry
Karolin Wellner, Mario Mörl
Since their initial discovery, tRNAs have risen from sole adapter molecules during protein synthesis to pivotal modulators of gene expression. Through their many interactions with tRNA-associated protein factors, they play a central role in maintaining cell homeostasis, especially regarding the fine-tuning in response to a rapidly changing cellular environment. Here, we provide a perspective on current tRNA topics with a spotlight on the regulation of post-transcriptional shaping of tRNA molecules. First, we give an update on aberrant structural features that a yet functional fraction of mitochondrial tRNAs can exhibit...
September 19, 2018: Biochemistry
Isabel Morgado, Afra Panahi, Andrew G Burwash, Madhurima Das, John E Straub, Olga Gursky
Hereditary apolipoprotein A-I (apoA-I) amyloidosis is a life-threatening incurable genetic disorder whose molecular underpinnings are unclear. In this disease, variant apoA-I, the major structural and functional protein of high-density lipoprotein, is released in a free form, undergoes an α-helix to intermolecular cross-β-sheet conversion along with a proteolytic cleavage, and is deposited as amyloid fibrils in various organs, which can cause organ damage and death. Glu34Lys is the only known charge inversion mutation in apoA-I that causes human amyloidosis...
September 19, 2018: Biochemistry
Derek F Harris, Zhi-Yong Yang, Dennis R Dean, Lance C Seefeldt, Brian M Hoffman
The enzyme nitrogenase catalyzes the reduction of N2 to ammonia but also that of protons to H2 . These reactions compete at the mechanistically central 'Janus' intermediate, denoted E4 (4H), which has accumulated 4e- /4H+ as two bridging Fe-H-Fe hydrides on the active-site cofactor. This state can lose e- /H+ by hydride protonolysis (HP) or become activated by reductive elimination ( re) of the two hydrides and bind N2 with H2 loss, yielding an E4 (2N2H) state that goes on to generate two NH3 molecules. Thus, E4 (4H) represents the key branch point for these competing reactions...
September 19, 2018: Biochemistry
Lorenza Favrot, Tathyana Amorim Franco, John S Blanchard
The biosynthesis of branched-chain amino acids or BCAAs (L-isoleucine, L-leucine, and L-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of L-isoleucine, L-leucine, and L-valine is very efficient, requiring only eight enzymes. Threonine deaminase (TD), a PLP-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of L-threonine (L-Thr) to -ketobutyrate, ammonia and water and is the first step in the biosynthesis of L-isoleucine...
September 18, 2018: Biochemistry
Shangyu Hong, Bo Zhai, Pavlos Pissios
Methyl donor balance is critical for epigenetic regulation in cells and is maintained by the so-called methionine cycle proteins that regenerate SAM, the universal methyl donor, from homocysteine formed by the activity of methyltransferases. Nnmt is a liver enzyme that methylates nicotinamide but its role in regulating methyl donor balance in the liver is unclear. In this study, we assessed the effect of altered Nnmt expression on various aspects of methyl donor metabolism in the liver. We found that Nnmt overexpression lowered SAM levels and SAM/SAH ratio in both in vivo and in vitro...
September 18, 2018: Biochemistry
John M Robbins, Jiafeng Geng, Bridgette A Barry, Giovanni Gadda, Andreas S Bommarius
Formate oxidase (FOX) was previously shown to contain a non-covalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and kinetic parameters previously reported for FOX are inconsistent with more recent reports. The UV-visible absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy (ICP-OES), UV-visible absorption spectroscopy, circular dichroism (CD) spectroscopy, liquid chromatography mass spectrometry (LC/MS), and electron paramagnetic resonance (EPR) spectroscopy...
September 18, 2018: Biochemistry
Erik Henrich, Frank Lőhr, Grzegorz Pawlik, Oliver Peetz, Volker Dötsch, Nina Morgner, Anton Ipm Kroon, Frank Bernhard
Biomembranes composed of lipids and proteins play central roles in physiological processes and the precise balance between different lipid species is crucial for maintaining membrane function. One pathway for the biosynthesis of the abundant lipid phosphatidylcholine in eukaryotes involves a membrane integrated phospholipid methyltransferase named Opi3 in yeast. A still unanswered question is whether Opi3 is able to catalyze phosphatidylcholine synthesis in trans, at membrane contact sites. While evidence for this activity was obtained from studies with complex in vitro reconstituted systems based on ER mem-branes, isolated and purified Opi3 could not be analyzed so far...
September 18, 2018: Biochemistry
Mingjie Li, Salette Martinez, Robert P Hausinger, Joseph P Emerson
The ethylene-forming enzyme (EFE), like many other 2-oxoglutarate (2OG)-dependent nonheme iron(II) oxygenases, catalyzes the oxidative decarboxylation of 2OG to succinate and CO2 to generate a highly reactive iron species that hydroxylates a specific alkane C-H bond, in this case targeting l-arginine (Arg) for hydroxylation. However, the prominently observed reactivity of EFE is the transformation of 2OG into ethylene and three molecules of CO2 . Crystallographic and biochemical studies have led to several proposed mechanisms for this 2-fold reactivity, but the detailed reaction steps are still obscure...
September 18, 2018: Biochemistry
Michael H Räz, Shana J Sturla, Hailey L Gahlon
Chemically-induced DNA lesions, persisting in the genome can become DNA replication substrates that are bypassed by low-fidelity DNA polymerases. Following incorporation of an incorrect base opposite a DNA lesion, the extension step can contribute to preserving such errors and lead to genomic instability and cancer. DNA polymerase ζ, a B-family polymerase, is uniquely proficient as an extender polymerase that catalyzes elongation, however, it is not understood what chemical factors impact DNA polymerase ζ-mediated DNA replication, especially past sites of DNA lesions...
September 17, 2018: Biochemistry
Jordon M Inloes, Hui Jing, Benjamin F Cravatt
Deleterious mutations in the serine hydrolase DDHD domain containing 1 (DDHD1) cause the SPG28 subtype of the neurological disease hereditary spastic paraplegia (HSP), which is characterized by axonal neuropathy and gait impairments. DDHD1 has been shown to display PLA1-type phospholipase activity with a preference for phosphatidic acid. However, the endogenous lipid pathways regulated by DDHD1 in vivo remain poorly understood. Here we use a combination of untargeted and targeted metabolomics to compare the lipid content of brain tissue from DDHD1+/+ and DDHD1-/- mice, revealing that DDHD1 inactivation causes a substantial decrease in the level of polyunsaturated lysophosphatidylinositol (LPI) lipids and a corresponding increase in the level of phosphatidylinositol (PI) lipids...
September 17, 2018: Biochemistry
Alison G Tebo, Frederico M Pimenta, Yu Zhang, Arnaud Gautier
Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens...
September 17, 2018: Biochemistry
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