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Sandip K Nandi, Stefan Rakete, Rooban B Nahomi, Cole Robert Michel, Alexandra Dunbar, Kristofer S Fritz, Ram H Nagaraj
Acylation of lysine residues is a common post-translational modification of cellular proteins. Here, we show that lysine succinylation, a type of acylation, occurs in human lens proteins. All the major crystallins exhibited Nε -succinyllysine (SuccK) residues. Quantification of SuccK in human lens proteins (from donors between the ages of 20 and 73 years) by LC-MS/MS showed a range between 1.2 and 14.3 pmol/mg lens protein. The total SuccK levels were slightly reduced in aged lenses (age > 60 years) relative to young lenses (age < 30 years)...
February 13, 2019: Biochemistry
Keya W Mukherjee, Jamison Huddleston, Tamari Narindoshvili, Venkatesh Nemmara, Frank M Raushel
A combination of bioinformatics, steady-state kinetics, and NMR spectroscopy has revealed the catalytic functions of YcjQ, YcjS and YcjR from the ycj gene cluster in Escherichia coli K-12. YcjS was determined to be a 3-keto-D-glucoside dehydrogenase with a kcat = 22 s-1, and kcat/Km = 2.3 x 104 M-1 s-1 for the reduction of α-methyl-3-keto-D-glucoside at pH 7.0 with NADH. YcjS also exhibited catalytic activity for the NAD+-dependent oxidation of D-glucose, β-methyl-D-glucoside, and 1,5-anhydro-D-glucitol. YcjQ was determined to be a 3-keto-D-guloside dehydrogenase with kcat = 18 s-1, and kcat/Km = 2...
February 11, 2019: Biochemistry
David F Savage
No abstract text is available yet for this article.
February 11, 2019: Biochemistry
Rebecca Feiner, Ina Pennè, Benjamin Müller, Kristian M Müller
The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth factor (EGF) with the fluorescent reporter mCherry can be expressed in the cytosol of E. coli in high yields and with high biological activity. Biophysical characterization by mass spectrometry analysis confirmed three disulfide bonds that are crucial for protein structure...
February 8, 2019: Biochemistry
Yifei Li, Olivier Soubias, Jess Li, Shangjin Sun, Paul A Randazzo, R Andrew Byrd
Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers...
February 8, 2019: Biochemistry
Andrew N Bigley, Dao Feng Xiang, Tamari Narindoshvili, Charlie W Burgert, Alvan C Hengge, Frank M Raushel
Organophosphorus flame-retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame-retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core...
February 7, 2019: Biochemistry
Samuel J Pellock, William G Walton, Matthew R Redinbo
The human gut microbiome is a ripe space for the discovery of new proteins and novel functions. Many genes in the gut microbiome encode glycoside hydrolases that help bacteria scavenge sugars present in the human gut. Glycoside hydrolase family 2 (GH2) is one group of sugar-scavenging proteins, which include β-glucuronidases (GUS) and β-galacturonidases (GalAses), enzymes that cleave the sugar conjugates of the epimers glucuronate and galacturonate. Here we structurally and functionally characterize a GH2 GalAse and a hybrid GUS/GalAse, which reveal the molecular details that enable these GHs to differentiate a single stereocenter...
February 7, 2019: Biochemistry
Deepika Prasad, Kalappa Muniyappa
Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. Further, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function...
February 6, 2019: Biochemistry
Matías D Hartman, Romina Inés Minen, Alberto A Iglesias, Carlos M Figueroa
Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than for NADP+ . Herein, we combined structural and kinetic data to reverse cofactor specificity of this enzyme. Using site-saturation mutagenesis we obtained the D216A mutant, which uses both NAD+ and NADP+ , although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1 , respectively)...
February 6, 2019: Biochemistry
Javier Navarro
Chemokines are important players in immune defense by directing migration of leukocytes, and as key promoters of tumorigenesis and metastasis. The present study explores the molecular mechanisms of recognition and activation of two homologous chemokine receptors CXCR1 and CXCR2, using CXCL8 analogs with residue substitutions in the conserved triad Glu4Leu5Arg6 (ELR). Analysis of the binding data of CXCL8 analogs to CXCR1 is consistent with the two-site model for signal recognition of CXCR1, whereas binding analysis of CXCL8 analogs to CXCR2 supported a single-site model for signal recognition of CXCR2...
February 6, 2019: Biochemistry
Dominic J Glover, Dawei Xu, Douglas S Clark
No abstract text is available yet for this article.
February 6, 2019: Biochemistry
Qian Chu, Alan Saghatelian
No abstract text is available yet for this article.
February 6, 2019: Biochemistry
Ethan D Evans, Zachary P Gates, Zhen-Yu J Sun, Alexander J Mijalis, Bradley L Pentelute
A 29-residue peptide (MP01) - identified by in vitro selection for reactivity with a small molecule perfluoroaromatic - was modified and characterized using experimental and computational techniques, with the goal of understanding the molecular basis of its reactivity. These studies identified a six amino acid point mutant (MP01-Gen4) that exhibited a reaction rate constant of 25.8 ± 1.8 M-1 s-1 at pH 7.4 and room temperature, approximately two orders of magnitude greater than its progenitor sequence and three orders of magnitude greater than background cysteine reactivity...
February 6, 2019: Biochemistry
Michael R Duff, Nidhi Desai, Michael A Craig, Pratul K Agarwal, Elizabeth E Howell
Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. Due to its role in one carbon metabolism, chromosomal DHFR is the target of the antibacterial drug, trimethoprim. Resistance to trimethoprim has resulted in a type II DHFR that is not structurally related to the chromosomal enzyme target. Because of its metabolic significance, understanding DHFR kinetics and ligand binding behavior in more cell-like conditions, where the total macromolecule concentration can be as great as 300 mg/mL, is important...
February 6, 2019: Biochemistry
Mathew Sebastiao, Noe Quittot, Isabelle Marcotte, Steve Bourgault
Amyloids are polypeptide supramolecular assemblies that have been historically associated with numerous pathologies. Nonetheless, recent studies have identified many amyloid structures that accomplish vital physiological functions. Interestingly, amyloid fibrils, either pathological or functional, have been reported to be consistently associated with other biomolecules such as RNA and glycosaminoglycans (GAGs). These linear polyanions, RNA and GAGs, have also demonstrated an inherent ability to accelerate or to promote amyloid formation...
February 5, 2019: Biochemistry
Michele Larocca, Fabrizia Foglia, Agostino Cilibrizzi
This study reports a general method to calculate dihedral angles (φ and ψ) of a given amino acid sequence, focusing on potential energy and torque moment concepts. By defining these physical measures in relation to the chemical interactions that occur on each single amino acid residue within a peptide, the folding process is analysed as the result of main mechanical forces (MMF) exerted in the specific amino acid chain of interest. As a proof of concept, Leu-enkephalin was initially used as a model peptide to carry out the theoretical study...
February 5, 2019: Biochemistry
Christophe V F P Laurent, Erik Breslmayr, Daniel Tunega, Roland Ludwig, Chris Oostenbrink
Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from cellobiose oxidation being shuttled from the CDH dehydrogenase domain to its cytochrome domain and then to the LPMO catalytic site. In the current work, we investigate the interaction of four Neurospora crassa LPMOs and five CDH cytochrome domains from different species using computational methods...
February 4, 2019: Biochemistry
Yuan Zhi, Tamari Narindoshvili, Lydia Bogomolnaya, Marissa Talamantes, Ahmed El Saadi, Helene Andrews-Polymenis, Frank M Raushel
Non-typhoidal Salmonella can colonize gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions (SGD) in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 (yfeJ) encodes one of these proteins and it is currently annotated as a Type I glutamine amidotransferase. STM2437 was purified to homogeneity and its catalytic properties with a wide range of γ-glutamyl derivatives were determined...
February 4, 2019: Biochemistry
Shuntaro Takahashi, Hiromichi Okura, Naoki Sugimoto
Since the origin of life on Earth, the role of carrying genetic information has been presumably transferred from RNA to DNA. At present, cellular environments are extremely dense, packed with co-solutes and macromolecules. Hence, the preference between RNA-dependent RNA and DNA polymerization may be affected by molecular crowding. In this study, we investigated both RNA-dependent RNA and DNA polymerizations by tC9Y polymerase ribozyme, T7 RNA polymerase (T7 RNAP), and Klenow fragment DNA polymerase (KF) in different molecular crowding conditions...
February 4, 2019: Biochemistry
Jolanta Krucinska, Eric Falcone, Heidi Erlandsen, Akram Hazeen, Michael Nicolo Lombardo, Alexavier Estrada, Victoria Lee Robinson, Amy Anderson, Dennis L Wright
Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation, RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activity of α-, β- and γ- substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring plays an important role in the biological activity of the investigated compounds...
February 4, 2019: Biochemistry
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