bluuebunny/crossref_metadata_2025_split huggingface dataset:
Initial Characterization of DszA, a Monooxygenase from the Rhodococcus Biodesulfurization Pathway
Authors: Truong Sharon , Sanchez Misael 
Published: June 2021

Sulfur dioxide is a pollutant found in our atmosphere that affects our health, environment, and economy. The most common source of sulfur dioxide pollution is the combustion of fossil fuels. To control pollution, it is important to remove sulfur from these fuels. The current sulfur removal method, hydrodesulfurization, is inefficient because it cannot remove sulfur from refractory organosulfur compounds. There are multiple bacterial species, including Rhodococcus erythropolis, that can remove sulfur from aromatic compounds, such as dibenzothiophene (DBT). DBT is a major organosulfur compound found in crude petroleum. The biodesulfurization pathway in Rhodococcus erythropolis can successfully remove sulfur from DBT to produce hydroxybiphenyl and sulfite. Our study focuses on an enzyme in this biodesulfurization pathway, the DBT sulfone monooxygenase, DszA that catalyzes the conversion of DBT sulfone to 2′‐hydroxybiphenyl 2‐sulfinate. Our goal was to obtain pure, active, and soluble protein for initial characterization of DszA. Thus far, we have determined the optimal parameters for expression of DszA by various expression and pH trials. The optimal temperature and pH for enzyme expression were 18°C and 7.5, respectively. DszA was successfully overexpressed in Escherichia coli and will be ready to purify by nickel affinity chromatography to take advantage of the His tag found in our protein. Our future steps are to apply steady‐state and transient‐kinetics to determine the kinetic parameters and study the mechanism of this important reaction step in the Rhodococcus biodesulfurization pathway. We will also analyze the substrate and product by high‐performance liquid chromatography to determine substrate specificity. These initial studies will provide a greater understanding towards the biodesulfurization mechanism for industrial purposes and pollution control.Support or Funding InformationThis research was supported by the Research Corporation (CCSA 22672) and by the National Institutes of Health (NIH) Grant 5SC2AI109500.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Iterative Methylations by a Cobalamin‐Dependent Radical SAM Methyltransferase
Authors: Wang Yuanyou , Begley Tadhg P. 
Published: June 2021

Natural products with branched alkoxy groups have been used widely in the development of bioactive compounds. However, the biosynthesis of branched alkoxy groups like the unique t‐butyl group is still poorly understood. The cystobactamids are a novel class of isopropyl substituted antibacterial compounds produced by myxobacteria. In the biosynthesis gene cluster of cystobactamid, CysS is assigned as a cobalamin‐dependent radical S‐adenosylmethionine (SAM) enzyme, which is proposed to catalyze the iterative methylations on the 3‐methoxy‐4‐aminobenzoic acid moieties of cystobactamids. To reconstitute CysS, different substrates analogs were synthesized. When the pantetheinyl substrate analogs were used, multiple methylations were observed to form ethyl, isopropyl, t‐butyl and sec‐butyl substituted products from a substrate methyl ether by CysS. These cobalamin dependent methylations also produce 5′‐deoxyadenosine(5′dA) and S‐adenosylhomocysteine (SAH) in equal amounts. The isotope labeling experiments suggest that the methyl group from SAM is first transferred to cobalamin followed by the attack of the substrate radical to give methylated products. A combinatorial antibiotic library of 25 cystobactamid analogs could potentially be built using CysS by Cystobacter sp. Different substituted substrate analogs were designed to trap the organic radical species, cobalamin intermediates and probe the radical substitution step. Here, we have showed the first in vitro reconstitution of a cobalamin‐dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t‐butyl group by sequential methylations. The detailed mechanistic studies help us to unravel how CysS uses SAM, [4Fe–4S] cluster and cobalamin to perform iterative methylations on sp3‐hybridized carbon centers and better understand the mechanism of other widespread cobalamin‐dependent radical SAM methyltransferases.Support or Funding InformationThis work was supported by NIH (DK44083) and by the Robert A. Welch Foundation (A‐0034).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective inhibition ATP Synthase in combating microbial infections
Authors: Ahmad Zulfiqar 
Published: June 2021

BackgroundATP synthase is main source of cellular energy for almost all organisms. Malfunction or inhibition of ATP synthase may cause cell death. A wide variety of inhibitors including phytochemicals and peptides are known to bind and inhibit ATP synthase. These phytochemicals and peptides bind to the specific binding pockets on ATP synthase. These binding pockets are flanked by variable amino acids in different species. Our lab is exploring the possibility of using ATP synthase as a selective molecular drug target to combat the antibiotic resistant microbial infections using Escherichia coli as a model organism.MethodWild type, null, and mutant E. coli growth properties were tested on fermentable glucose and non‐fermentable succinate carbon sources. Wild type and mutant enzymes were isolated by harvesting cells in the minimal media. Inhibitory studies were performed on membrane bound F1Fo ATP synthase. Structural modifications of inhibitors were made through replacement or re‐positioning of the functional groups on phytochemicals or addition of positive charges on the peptides. Wild type and mutant cell growth assays were tested in presence and absence of inhibitors along with null control.ResultsPhytochemicals and peptides caused variable degree of inhibition. Extent of inhibition was augmented by modification of inhibitors. For phytochemicals, re‐positioning and addition of new functional groups and for peptides, addition of a c‐terminal NH2 group enhanced the inhibitory potency on molar scale. We also found that incremental addition of positively charged residues in peptides can augment the inhibitory effects of peptides by about 100‐fold. Cell growth assays in presence and absence inhibitors suggest that ATP synthase is a potent molecular drug target to combat microbial infections. Currently, we are studying the synergistic inhibitory effects of phytochemicals and peptides on ATP synthase.ConclusionsWe conclude that phytochemicals and peptides bind and inhibit E. coli by interacting at the specific sites on F1 sector of ATP synthase and ATP synthase is a potential molecular drug target.Support or Funding InformationKCOM‐ATSU Biomedical Sciences Graduate Program Grant Award # 850‐611, 850‐619, and 851‐026This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IDH1 mutation‐inspired α‐ketoglutaric acid mimics for oxidative therapy of higher grade gliomas through α‐ketoglutarate dehydrogenase inhibition
Authors: Madala Hanumantha Rao , Punganuru Surendra Reddy , Srivenugopal Kalkunte 
Published: June 2021

Mutations at the active site of isocitrate dehydrogenase 1 gene (IDH1; R132H) occur at a high frequency (>70%) early in the oncogenesis of lower grade malignant gliomas, and result in a dramatic accumulation of the oncometabolite D‐2 hydroxyglutarate (D‐2HG), that effectively replaces the normal metabolite α‐ketoglutarate (α‐KG) in cell physiology. Surprisingly, IDH1 mutations bestow superior therapeutic responses to alkylating agents and better patient survival. D‐2HG, effectively competes with α‐KG and potently inhibits various dioxygenase reactions, including the TET1&2 DNA ‐demethylases and histone demethylases thereby, re‐shaping and reprogramming the epigenetic landscape, and consequent transcriptional silencing; the DNA repair protein O6‐lguanine DNA methyltransferase (MGMT), which confers drug resistance is one major target for such repression. As a novel and innovative strategy of turning the tide against GBMs by exploiting the mechanistic aspects of the oncometabolite, we hypothesized that D‐2HG and α‐KG derivatives that can replace the natural metabolite in epigenomic dioxygenase reactions will serve as potent anti‐glioma drugs either by themselves or in combination with the alkylating agents. To this end, we synthesized a 2,4‐dimethyleneglutaric acid (DMG), an α‐KG mimic with methylene groups inserted at the C2 and C4 positions. The hydrophobic DMG ester, by itself, was cytotoxic with IC50 values up to 300 μM against brain tumor cell lines, however, when combined at 100 μM with TMZ resulted in >20‐fold increased synergistic cell killing. DMG induced oxidative stress inhibited α‐KG dehydrogenase, MGMT and TET1&2. DMG induced oxidative stress further caused mitochondrial damage as confirmed with JC‐1 staining. Mitochondrial damage further affected its biosynthetic, bioenergetic and signaling functions as confirmed with respective assays. We also observed a marked reduction in ATP and NADH levels and rapid metabolic flux changes indicating an altered cellular metabolism. Metabolomic analysis of major Krebs cycle intermediates revealed significant decrease in their levels. We, further observed that, bioenergetic stress induced by DMG activated AMPK and inhibited mTOR signaling cascade. Sub‐acute toxicity on mice revealed the safety of DMG in mice and we observed significant regression in tumor growth in intracranial xenograft tumor models. Collectively, these data reveal that acute treatments of α‐KG analogs can alter the cellular metabolic and epigenetic makeup in a manner ascribed to D‐2HG, and open up much‐needed novel and exciting avenues of oncometabolite‐based therapies for brain tumorsSupport or Funding InformationCPRIT grants RP130266 and RP170207 to KSSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Analysis of Darunavir Analogs Against Primary Resistance Mutations in HIV Protease
Authors: Lockbaum Gordon Joseph , Leidner Florian , Rusere Linah , Nalivaika Ellen , Ali Akbar , Kurt‐Yilmaz Nese , Schiffer Celia 
Published: June 2021

Antiretroviral therapies (ARTs) involve combinations of small molecule inhibitors that suppress the viral load of Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Symptom (AIDS). ARTs, currently the only defense against HIV, target critical proteins necessary for viral replication, such as HIV‐1 protease. The most potent FDA approved protease inhibitor, darunavir (DRV), is still susceptible to resistance if the protease acquires mutations in and around the active site. One method to avoid resistance is to optimize the inhibitor to fit the consensus volume that the protease's natural substrates occupy. Using this strategy, DRV analogs with larger moieties at the P1’ position were designed and synthesized to leverage unexploited space and create additional protease‐inhibitor interactions, resulting in inhibitors that are more potent and less susceptible to resistance. To elucidate the molecular mechanisms of active site resistance mutations against these inhibitors, we used a multidisciplinary approach, including biochemical assays and x‐ray crystallography. Results suggest that modifications to the DRV scaffold result in inhibitors with potency comparable to DRV, but structural analysis highlights differences in intermolecular and intramolecular interactions, which will inform future drug design.Support or Funding InformationThis work was supported in part by a grant from the National Institute of General Medical Sciences, National Institutes of Health (P01‐GM109767).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and Purification of Hydrophobic PEP‐Inhibiting Peptides from Bovine αS1 Casein
Authors: Sheffield Logan E. , Whalen Kaylei D. , Goode Courtney A. , Acosta Francisco M. , Lenert Melissa E. , Clack Beatrice A. 
Published: June 2021

According to previous studies, there is a prolyl endopeptidase‐inhibiting sequence (LNENLLRFFVAPFPEVFG) within a relatively hydrophobic region of αS1 casein (Juillerat‐Jeanneret et al., 2011). Recently, sixteen recombinant peptides were designed in assorted lengths, each containing the inhibitory sequence at varied positions within the αS1 casein sequence. The corresponding cDNA sequences were originally ligated into the pET15b vector, which provides an N‐terminal poly‐histidine tag for purification. The vector was in turn transformed into a BL21(DE3)pLysS E. coli expression host (Vishram et al., 2016). However, the system proved unsuccessful in adequate expression of the hydrophobic peptide. The intention of this study was to assemble a new pET28a expression system, kindly provided by Dr. X. Cheng (MD Anderson in Houston, TX) that utilizes a SUMO (small ubiquitin‐like modifier) fusion tag. The SUMO tag is known for enhancing expression levels of potentially toxic or insoluble recombinant peptides (Bommarius et al., 2010). The expressed peptides were purified via Ni‐NTA column chromatography, with subsequent UlpI cleavage. This was followed by reversed‐phase high performance liquid chromatography (RP‐HPLC) to further purify the peptides. Tricine‐SDS‐PAGE, a useful screening tool for hydrophobic peptides smaller than 30kDa, was used to analyze expression and purification. A comparison was made between SUMO‐fused expression and non‐fused expression of the hydrophobic casein peptides.Support or Funding InformationOffice of Research and Sponsored Projects, SFASU and Ed & Gwen ColeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Covalent attachment of protein cargo to a computationally designed toroid scaffold
Authors: Polkinghorn Caley Ray , Hallinan Jazmine , Doyle Lindsey , Bradley Phil , Stoddard Barry , Kaiser Brett K. 
Published: June 2021

Recent developments in computational design have expanded the accessible protein structures to those beyond what was selected for through the evolutionary process. Closed alpha‐solenoid tandem repeat structures (alpha‐toroids) are one class of rationally designed proteins. Four designed toroids, each containing a central pore and a varied number of repeats, were previously validated by x‐ray crystallography. We envision using toroids as stabilizing scaffolds upon which other proteins can be attached. The goal of this study is to develop an approach for attaching protein cargo to the toroid using the Spycatcher/Spytag peptide tagging system. Spycatcher (protein) and Spytag (peptide) are split protein partners derived from the fibronectin binding protein of Streptococcus pyogenes that form an irreversible, stable isopeptide bond. We demonstrated that 24‐repeat toroids containing four Spycatcher domains embedded within loop regions (“toroid‐SpyCatcher”) properly fold and assemble by size exclusion chromatography. We then showed that a Spytag‐GFP fusion protein binds efficiently and irreversibly to the toroid‐SpyCatcher using fluorescent binding assays. This proof of principle experiment demonstrates that this approach is feasible for attaching proteins to the toroid scaffold and could be expanded to attach cargo proteins with diverse activities.Support or Funding InformationMurdock Charitable TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational design and molecular mechanism in oligomerization of C‐terminal binding protein 2
Authors: Miyashita Yurina , Matsuo Naoya , Yamamoto Yuta , Nakano Shogo , Ito Sohei , Numoto Nobutaka , Ikura Teikichi , Ito Nobutoshi , Sekiya Motohiro , Shimano Hitoshi , Tokiwa Hiroaki 
Published: June 2021

IntroductionC‐terminal binding protein 2 (CtBP2) is moonlighting proteins, which has multiple functions and plays a key role in transcriptional control and in the maintenance of cellular metabolism (1). CtBP2 consist of D‐isomer specific 2‐hydroxyacid dehydrogenase (D2‐HDH) domains and C‐terminal intrinsically unstructured region. Recent studies have shown that the recruitment of a corepressor complex by CtBP2 depends on NADH/NAD ratio. Molecular mechanism of oligomerization of CtBP2 may provide a basis for the formation of a transcriptional corepressor complex.MethodsWe attempted to construct artificial designed proteins on the basis of in silico screening using the INTMSAlign program (2). Overexpression in E. coli cells, and purification of artificial designed CtBP2 (ArCtBP2) were performed. The oligomeric state and the binding affinity to NADH of ArCtBP2 were compared to human CtBP2 (hCtBP2) by size exclusion chromatography and intrinsic Trp fluorescence. To evaluate structural information, X‐ray crystallographic analysis of ArCtBP2 was performed.ResultsSize exclusion chromatography of ArCtBP2 shows 4 peaks for each corresponding oligomeric state, whereas that of hCtBP2 was observed as a single peak. The dissociation constants (Kd) value of NADH to ArCtBP2 was determined as 3.6 ± 0.9 μM by fluorescence spectroscopy. In addition, crystal structure of apo and NAD–bound form of ArCtBP2 at 2.0 and 1.7 Å resolution, respectively. Crystal structure of ArCtBP2 showed opened‐like structure in D2‐HDH domain compared to hCtBP2. These results suggested that the open–closed form of D2‐HDH domain is involved in the binding affinity for NADH and oligomeric state of CtBP2.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of New Molecular Tools for Antibody Detection and Purification
Authors: de Lourdes Borba Magalhaes MAria , da Silva Gustavo Felippe , Echeverri Lina Maria Salazar , Fernandes Leonardo Antonio , Batista Franciane 
Published: June 2021

Primary antibody detection is crucial for biomarker detection in research and diagnosis. Molecular tools for antibody detection and purification currently available consist of secondary antibodies as well as bacterial proteins such as Proteins A and G. Disadvantages of the current methods include the fact that secondary antibody production is expensive and may require the use of animals. In the case of Proteins A and G, they either do not recognize all IgG isotypes or require acidic elution conditions for antibody purification, which may lead to antibody denaturation. We have produced chimeric proteins, using an IgG binding protein recently discovered (TRIM21) linked to detection proteins such as streptavidin, and alkaline phosphatase. TRIM21 is a cytosolic protein that binds all IgG isotypes from many species. Bacterial expression, purification and refolding of TRIM21‐streptavidin as well as TRIM21‐alkaline phosphatase were successful. TRIM21‐streptavidin chimeric protein was incubated with biotinylated HRP or alkaline phosphatase for immunoassays. ELISA and western blotting experiments demonstrated that the chimeric proteins efficiently detect monoclonal and polyclonal antibodies from a wide range of species, including human, mouse, rat, dog, horse and bovine. Binding experiments are currently underway to determine the binding constants for TRIM21 chimeric proteins and IgG from different species. Future experiments will be performed using imobilized TRIM21 for antibody purification.Support or Funding InformationThis work was supported by the “ State University of Santa Catarina” (UDESC), “Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina” (FAPESC) and CNPQ.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How to design artificial protein surpassing native enzyme function ~ Design and multidisciplinary analysis of artificial L‐threonine 3‐dehydrogenase ~
Authors: Nakano Shogo , Motoyama Tomoharu , Miyashita Yurina , Ishizuka Yuki , Matsuo Naoya , Tokiwa Hiroaki , Shinoda Suguru , Asano Yasuhisa , Ito Sohei 
Published: June 2021

IntroductionCurrently, we can easily obtain a large amount of protein sequence data through database, such as PubMed. The data are being used for protein engineering to reconstruct sequence of target protein, like full‐consensus protein design (FPD) and ancestral sequence reconstruction (ASR). However, redesign of highly functional protein is challenging tasks. In this study, we attempt to get a clue how to succeed in the redesign through protein sequence reconstruction of native l‐threonine 3‐dehydrogenase from Cupriavidus necator (CnTDH) (1).MethodsFirstly, total 5000 of homologous sequences with CnTDH were obtained by submitting sequence of CnTDH to Blastp: the sequences were called “library”. Artificial TDH, FpdTDH‐N0, was designed by the FPD method using the library. Next, utilizing INTMSAlign (2, 3), the curated library was generated to maximize frequency of each consensus residues of artificial TDH. Two artificial TDHs were designed by FPD (FpdTDH‐N1) and ASR (AsrTDH) method, respectively, utilizing the curated library.Results & DiscussionSpecific activity was measured for the three artificial TDHs; and the activity could be confirmed for FpdTDH‐N1 and AsrTDH which designed using the curated library. FpdTDH‐N1 and AsrTDH share 69 and 65% of sequence identity with CnTDH, respectively. Enzyme properties of CnTDH, FpdTDH‐N1 and AsrTDH were estimated by the following multidisciplinary analysis. Thermal stability of both FpdTDH‐N1 and AsrTDH was better than that of CnTDH: Tm values of FpdTDH‐N1 and AsrTDH were higher about 10 and 5 °C than that of CnTDH. Enzyme kinetics parameters of FpdTDH‐N1 and AsrTDH (kcat/Km value) were comparable to CnTDH. Furthermore, dissociation constant (Kd) for AsrTDH and FpdTDH‐N1 toward NAD+ which measured by ITC were about 8‐ and 2‐fold lower than that of CnTDH. Structural comparison among CnTDH, FpdTDH‐N1 (2.9 Å resolution) and ArsTDH (2.2 Å resolution) indicated that there is no mutation around 7 Å of oxygen atom of catalytic Tyr. The mutation was assembled at NAD+ binding site and protein surface.Taken together, we succeeded in design artificial TDHs bearing high stability, activity and affinity toward NAD+ compared with native TDH, CnTDH. This result also inferred that curated library designed by maximizing frequency of amino acids for consensus residues of target protein is effective to design “super enzyme” surpassing native enzyme function.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number 16K18688, 17KT0010, 17H06169 and JST by ERATO grant number JPMJER1102.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.