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Results 1781 through 1800 out of 1950

in category biochemistry


1781: Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

Kristina Haslinger, Kristala L.J. Prather

110 downloads (posted 22 Jul 2019)

Background: Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far. Results: The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 μM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even an initially unproductive Cytochrome P450/ redox system combination productive. Conclusions: The caffeic acid titer achieved in this study is about 25% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.


1782: m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB

Chia-chieh Chu, Bei Liu et al.

110 downloads (posted 24 Oct 2019)

N 6 -methyladenosine (m 6 A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs.  m 6 A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions.  Two m 6 A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export.  However, because these m 6 A sites have not been observed in other studies mapping m 6 A in HIV-1 RNA, their significanc...


1783: Dissection of Catalytic Site in Crucial Gut Microbiome Enzyme: Bile Salt Hydrolase

Yashpal Yadav, Mrityunjay K. Tiwari et al.

109 downloads (posted 25 Jul 2019)

Bile Salt Hydrolases (BSHs) are enzymes from enteric bacteria that catalyze the hydrolysis of Bile Acids and consequently promote the reduction of cholesterol level in the mammalian body. Out of several reported BSHs, the Enterococcus faecalis BSH (EfBSH) has been reported to have the highest enzymatic activity. Herein, we have investigated the mechanistic details of the EfBSH activity. The study was carried out employing two mutants of EfBSH: E269A and R207A, which shows differential catalytic activity. The mutant E269...


1784: The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

Athanassios Adamopoulos, Tatjana Heidebrecht et al.

109 downloads (posted 20 Dec 2018)

JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tig...


1785: Broad spectrum antibiotic-degrading metallo-β-lactamases are phylogenetically diverse and widespread in the environment.

Marcelo Monteiro Pedroso, David Waite et al.

108 downloads (posted 16 Aug 2019)

Antibiotic resistance has emerged as a major global health threat. The Zn2+-dependent metallo-β-lactamases (MBLs) are of particular concern as they act on the most widely prescribed class of antibiotics, the β-lactams, and are largely unaffected by commonly used β-lactamase antagonists such as clavulanic acid. MBLs are subdivided into three groups (B1 to B3); despite low overall sequence similarity, their catalytic centers are conserved with two closely spaced Zn2+ binding sites (α and β site). We recovered almost 1500 ...


1786: Elevated Expression of a Functional Suf Pathway in the E. coli BL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein.

Elliot I Corless, Erin L Mettert et al.

108 downloads (posted 18 Oct 2019)

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes withi...


1787: Non-additive effects of changing the cytochrome P450 ensemble: incorporation of CYP2E1 into human liver microsomes and its impact on CYP1A2

Nadezhda Y Davydova, Bikash Dangi et al.

108 downloads (posted 27 Jun 2019)

The aim of this study is to investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system we used CYP2E1-enriched microsomes obtained by incorporation of purified CYP2E1 protein into HLM. Using the method based on homo-FRET in homo-oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with microsomes result in dissociation of the protein homo-oligomers. The finding tha...


1788: Divergent interactions maintain the quaternary octameric structure of a new family of esterases

Onit Alalouf, Rachel Salama et al.

108 downloads (posted 19 Nov 2018)

Protein oligomerization contributes significantly to the stability and function of enzymes, and the interacting interfaces that create the oligomers are expected to be conserved. The acetyl-xylo-oligosaccharide esterase, Axe2, from the thermophilic bacterium Geobacillus stearothermophilus represents a new family of esterases belonging to the SGNH superfamily of hydrolytic enzymes, and has a unique doughnut-like homo-octameric configuration, composed of four homo-dimers. The dimers of Axe2 are held together mainly by clu...


1789: MYPT1 O-GlcNAcylation Dictates Timely Disjunction of Centrosomes

Caifei Liu, Yingxin Shi et al.

107 downloads (posted 07 Oct 2019)

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruption have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here we attempted to address the underlying molecular mechanism by both chemical approaches and biological assays, and observed that Thiamet-G (OGA inhibitor...


1790: Ginsenoside Rg1 defenses PC-12 cells against hydrogen peroxide-caused damage via up-regulation of miR-216a-5p

Guangkun Yi, Li Liu et al.

107 downloads (posted 06 Jun 2019)

Background: Spinal cord injury (SCI) is a destructive trauma accompanying with local injury, half of which cause chronic paralysis. Ginsenoside Rg1 exerts anti-apoptosis and anti-autophagy properties. Therefore, our goal was to study the protective mechanism of Rg1 in attenuating cell injury. Methods: MiR-216a-5p inhibitor was transfected into PC-12 cells, then cells were pre-treated by Rg1 and treated with 300 μM hydrogen peroxide (H2O2) for 24 h. CCK-8 and apoptosis experiments were done to test cell activity and apop...


1791: DSCAM-AS1 promotes tumor growth of breast cancer by reducing miR-204-5p and upregulating RRM2

Wen-Hui Liang, Na Li et al.

107 downloads (posted 27 Aug 2018)

Purpose: We intended to analyze the effects of DSCAM-AS1, miR-204-5p and RRM2 on breast cancer (BC) cells growth. Methods: Microarray analysis and qRT-PCR were employed to determine DSCAM-AS1 and miR-204-5p expression in BC. Luciferase reporter assay and cell transfection assay were applied to examine the target relationship between DSCAM-AS1, miR-204-5p and MMR2. CCK-8 assay, transwell assay and flow cytometry were used to detect cell proliferation, invasion and apoptosis of breast cancer cells. The expression of DSCAM...


1792: Thrombospondin module 1 domain (TSP1) of the matricellular protein CCN3 shows an atypical disulfide pattern and incomplete CWR layers

Emma-Ruoqi Xu, Aleix Lafita et al.

106 downloads (posted 23 Sep 2019)

Members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins, essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of its four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis, and tumorigenesis. We present here the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously...


1793: Compound heterozygosity of the gamma-glutamyl carboxylase mutants V255M and S300F causes pseudoxanthoma elasticum-like disease through impaired processivity

Mark A. Rishavy, Kevin W. Hallgren et al.

105 downloads (posted 12 Jun 2019)

Vitamin K-dependent (VKD) protein activities require carboxylated Glus (Glas) generated by the gamma-glutamyl carboxylase. Some carboxylase mutations cause severe bleeding, while others cause pseudoxanthoma elasticum (PXE)-like associated with excessive calcification. How carboxylase mutations cause PXE-like was unknown. We analyzed two mutants (V255M and S300F) whose compound heterozygosity causes PXE-like. Substrates derived from VKD proteins important to calcification (MGP) or clotting (factor IX) were studied, which...


1794: Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3

Matthias Romauch

104 downloads (posted 07 Aug 2019)

Zinc-alpha2-glycoprotein (ZAG) is a major plasma protein whose levels increase in chronic energy-demanding diseases and thus serves as an important clinical biomarker in the diagnosis and prognosis of the development of cachexia. Current knowledge suggests that ZAG mediates progressive weight loss through beta-adrenergic signaling in adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through crosslinking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a novel ZAG bind...


1795: Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics

Aviv Meir, Veronica Lepechkin-Zilbermintz et al.

104 downloads (posted 09 Sep 2019)

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living...


1796: Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors

Anubha Seth, Manuel Finkbeiner et al.

103 downloads (posted 06 Jul 2019)

Mammalian adenylate cyclases (ACs) are pseudoheterodimers with dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. We exchanged both membrane anchors of the AC isoform 2 by the isosteric quorum-sensing receptor from Vibrio , CqsS, which has a ligand, Cholera -Autoinducer-1 (CAI-1). In the chimera, AC activity was stimulated by Gsα, CAI-1 had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation. We report that Gsα stimulation of human AC isoforms 2, 3, 5, and 9 ...


1797: The nuclear transport receptor TNPO1 binds macrophage immunometabolism regulator MACIR via a PY-NLS motif

Gavin McGauran, Emma Dorris et al.

103 downloads (posted 14 Nov 2019)

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and the regulation of macrophage biology through unknown mechanisms. The 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. Here we identify specific interactions of MACIR using a fragment complementation-based affinity pull down of cellular proteins prepared with a membrane solubilization buffer...


1798: Unprocessed serum glycosylphosphatidylinositol-anchored proteins are correlated to metabolic states

Günter A. Müller, Andreas W. Herling et al.

103 downloads (posted 28 Nov 2018)

To study the possibility that components of eukaryotic plasma membranes are released in spontaneous or controlled fashion, a chip-based sensor was developed for complete glycosylphosphatidylinositol-anchored proteins (GPI-AP), which may form together with (phospho)lipids so far unknown (non-vesicular) extracellular complexes (GLEC). The sensor relies on changes in phase shift and amplitude of surface acoustic waves propagating over the chip surface upon specific capturing of the GPI-AP and detection of associated phosph...


1799: Characterization of Sorbitol Dehydrogenase SmoS from Sinorhizobium meliloti 1021

MacLean G. Kohlmeier, Ben A. Bailey-Elkin et al.

102 downloads (posted 01 Jul 2019)

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+ as a cofactor. The main objective of this study i...


1800: Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions

Paula A Agudelo Garcia, Prabakaran Nagarajan et al.

102 downloads (posted 31 Oct 2019)

Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1+/+ and Hat1−/− mouse embryonic fibroblast (MEF) cells lines were grown in both glucose- a...