Synthesis and Characterization of photoactivatable doxycycline analogues bearing two-photon sensitive photoremovable groups suitable for light induced gene expression ChemBioChem (IF 2.847) Pub Date : 2018-01-17 Alexandre Specht, Bastien Goegan, Firat terzi, Frédéric Bolze, Sidney Cambridge
We report herein the synthesis and the photolytic properties of EANBP and PEG7-DEACM caged 9-aminodoxycycline. 9-Aminodoxycycline is a tetracycline analogue able to activate transcription via the inducible TetOn transgene expression system and can be regioselectively coupled by carbamoylation to two-photon sensitive photoremovable protecting groups. The EANBP caged 9-aminodoxycycline showed complex photochemical reactions but did release 10% of 9-aminodoxycycline. However, the PEG7-DEACM-9-aminodoxycycline exhibited excellent photolysis efficiency at 405 nm with a quantitative release of 9-aminodoxycycline and a 0.21 uncaging quantum yield. Due to the good two-photon sensitivity of the DEACM chromophore, 9-aminodoxycyline release is possible by two-photon photolysis with a calculated action cross-sections up to 4.0 GM at 740 nm. Therefore, PEG7-DEACM-9-aminodoxycycline represents a very attractive tool for the development of a light induced gene expression method in living cells.
Control of protein activity and gene expression by cyclofen-OH uncaging ChemBioChem (IF 2.847) Pub Date : 2018-01-16 Weiting Zhang, Fatima Hamouri, Zhiping Feng, Isabelle Aujard, Bertrand Ducos, Shixin Ye, Shimon Weiss, Michel Volovitch, Sophie Vriz, Ludovic Jullien, David Bensimon
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activable protein and the protein of interest to control the activity of the later, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. We here present that approach and its uses in a variety of physiological contexts.
The Fourth Wave of Biocatalysis Emerges—The 13 th International Symposium on Biocatalysis and Biotransformations ChemBioChem (IF 2.847) Pub Date : 2018-01-16 László Poppe, Beáta G. Vértessy
Efficient catalytic oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid by magnetic laccase catalyst ChemBioChem (IF 2.847) Pub Date : 2018-01-15 Ke-Feng Wang, Chun-lei Liu, Kun-yan Sui, Chen Guo, Chun-Zhao Liu
2,5-Furandicarboxylic acid (FDCA) is a biobased platform chemical for the production of polyesters. The magnetic laccase catalyst with TEMPO as the mediator has the remarkable capability of oxidizing HMF to 2,5-furandicarboxylic acid (FDCA). Under optimal reaction conditions, a quantitative yield (90.2%) of FDCA with complete HMF conversion was obtained after 96 h of reaction. More importantly, the magnetic laccase catalyst exhibited good recyclability and stability, maintaining 84.8% of its original activity following 6 reuse cycles. Herein, this is the first report on the efficient catalytic oxidation of HMF to FDCA using an immobilized enzyme (magnetic laccase catalyst).
Investigating the accessibility of nucleosome by duocarmycin B2, Fe(II) peplomycin and MNase using capillary electrophoresis ChemBioChem (IF 2.847) Pub Date : 2018-01-15 Tingting Zou, Seiichiro Kizaki, Hiroshi Sugiyama
Capillary electrophoresis coupled with DNA 5 Texas Red labeling was used to investigate the ability of duocarmycin B2, Fe(II) peplomycin, and MNase to access the nucleosome. Distinct accessibility patterns of these species to the nucleosome were observed. Duocarmycin B2 was able to enter and react at the core region, although its alkylating efficiency decreased significantly. Intercalation of peplomycin in the nucleosomal core region was highly suppressed, but reaction sites located at the ends of the nucleosomal core remained accessible, which implied the flexibility of the core DNA end. MNase was completely prevented from approaching the nucleosome core and exhibited a higher site specificity for targeting DNA sites located close to the core region.
Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging ChemBioChem (IF 2.847) Pub Date : 2018-01-15 Min Shen, Amit A. Joshi, Raghu Vannam, Chandra K. Dixit, Robert G. Hamilton, Challa V. Kumar, James F. Rusling, Mark W. Peczuh
Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases ChemBioChem (IF 2.847) Pub Date : 2018-01-14 Tobias Betke, Jun Higuchi, Philipp Rommelmann, Keiko Oike, Taiji Nomura, Yasuo Kato, Yasuhisa Asano, Harald Gröger
Nitriles belong to the product classes, which are mostly needed and produced by the chemical industry, playing a major role in various industry segments ranging from high-volume low-price sectors such as polymers to low-volume high-price sectors such as the chiral pharma drugs. A common industrial technology for nitrile production is ammonoxidation as a gas phase reaction at high temperature. A further popular approach are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed by means of so-called aldoxime dehydratases, which are capable to convert an aldoxime in one step via dehydration into nitriles. Since the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria to be applied as a "synthetic platform technology". Thus, the scope of this review is to give an overview about the current state of the substrate scope of this enzymatic method for synthesizing nitriles by means of aldoxime dehydratases. Even being a recently emerged enzyme class, a range of substrates have already been studied so far, comprising non-chiral and chiral aldoximes. It turned out that this enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes as well as arylaliphatic aldoximes. Furthermore, aldoximes bearing a stereogenic center are recognized as well and in particular for 2-arylpropylaldoximes high enantioselectivities are found. It is further noteworthy that the enantiopreference turned out to depend on the (E)- and (Z)-isomer. Thus, opposite enantiomers are accessible although starting from the same racemic aldehyde and the same enzyme.
Ketonization of proline residues in the peptide chains of actinomycins by a 4-Oxoproline synthase ChemBioChem (IF 2.847) Pub Date : 2018-01-12 Siamak Semsary, Ivana Crnovčić, Ronja Driller, Joachim Vater, Bernhard Loll, Ullrich Keller
X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X0) or 4-oxoproline (Acm X2) in their ß-pentapeptide lactone rings whereas their α-ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxo-proline-containing Acm halves. In turn we show - using an artificial Acm half analogue (PPL 1) with proline in its peptide chain - its conversion into the 4-hydroxyproline and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase and a ferredoxin, were identified. After co-expression in Escherichia coli their gene products converted PPL 1 into PPL 1 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner.saAcmM has high sequence similarity to the Cyp107Z (Ema) family of cytochrome P450s monooxygenases which can convert avermectin B1 into its keto derivative 4''-oxo-avermectin B1. Determination of the structure of saAcmM revealed high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylproline-ketonizing Cyps.
Modulation of BACE1 activity by chemically modified aptamers ChemBioChem (IF 2.847) Pub Date : 2018-01-11 Cécile Gasse, Marwa Zaarour, Sam Noppen, Mikhail Abramov, Philippe Marlière, Sandra Liekens, Bart De Strooper, Piet Herdewijn
A modified DNA aptamer was developed that binds BACE1, a therapeutic target involved in Alzheimer's disease. This ssXNA not only tightly binds to BACE1 but also inhibits its protease activity in vitro in the same range as a previously described unmodified aptamer. We report the in vitro selection of functional oligonucleotides using two nucleobase modifications: 5-chlorouracil and 7-deazaadenine. The nucleoside analog 5-chloro-2'-deoxyuridine has already been explored to replace thymidine in a chemically modified genome of a bacterium. Thus, 5-chlorouracil modification is a good candidate to support genetic transfer in vivo as well as functional activity.
Alphanumerical Visual Display Made of DNA Logic Gates for Drug Susceptibility Testing of Pathogens ChemBioChem (IF 2.847) Pub Date : 2018-01-12 Ryan P. Connelly, Evgeny S. Morozkin, Yulia V. Gerasimova
Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction ChemBioChem (IF 2.847) Pub Date : 2018-01-12 Aitao Li, Carlos G. Acevedo-Rocha, Zhoutong Sun, Tony Cox, Jia Lucy Xu, Manfred T. Reetz
RNA-Templated Concatenation of Triplet Nucleic Acid Probe ChemBioChem (IF 2.847) Pub Date : 2018-01-11 Danith H Ly, Raman Bahal, Arunava Manna, Wei-Che Hsieh, Shivaji A Thadke, Gopalsamy Sureshkumar
Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. While it is central to many biological processes, such an approach has rarely been applied to the in-situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic acid probe containing C-terminal thioester and N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of RNA target, and self-deactivation in its absence. The work has an implication for the development of millamolecular nucleic acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders.
A New Fluorogenic Probe for the Detection of Endo-β-N-acetylglucosaminidase ChemBioChem (IF 2.847) Pub Date : 2018-01-11 Nozomi Ishii, Chie Sunaga, Kanae Sano, Chengcheng Huang, Kenta Iino, Yuji Matsuzaki, Tadashi Suzuki, Ichiro Matsuo
We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-β-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase of fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase, which could serve as therapeutic agents for the treatment of NGLY1-deficiency.
A T7 RNA Polymerase Mutant Enhances the Yield of 5′-Thienoguanosine-Initiated RNAs ChemBioChem (IF 2.847) Pub Date : 2018-01-09 Seth Lyon, Venkat Gopalan
Analysis of quaternary structure of Hb Beckman variant and molecular interpretation of its functional abnormality: A mass spectrometry based approach ChemBioChem (IF 2.847) Pub Date : 2018-01-08 Monita Muralidharan, Rajdeep Das, Vijay Bhat, Amit Kumar Mandal
Electrostatic attraction between α and β globin chains hold the subunits together in tetrameric human hemoglobin molecule (α₂β₂). Compared to normal globin chains, the affinity of a mutant chain to its partner globin in genetic variants of hemoglobin might be different. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous sample, even though the rates of synthesis of both normal and variant chains are same. The aforementioned affinities across various globin chains might be assessed by the quantification of different forms of tetramers present in a variant hemoglobin sample.In the present study, exploiting mass differences between globin chains, differentially populated hemoglobin tetramers present in Hb Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α₂β₂, α₂ββV and α₂βV₂) indicated that both β and βV have differential affinities towards α globin chain. The conformational dynamics analyzed from hydrogen/deuterium exchange kinetics, of the signature peptide fragment of Hb Beckman in its oxy state, displayed molecular insights into its functional abnormality. However, in comparison to normal hemoglobin (α₂β₂), the point mutation did not show any change in the collision cross-sections of the functionally active conformers of the variant hemoglobin molecules (α₂ββV and α₂βV₂).
Identification of the biosynthetic gene cluster for himeic acid A, a ubiquitin-activating enzyme (E1) inhibitor, in Aspergillus japonicus MF275 ChemBioChem (IF 2.847) Pub Date : 2018-01-05 Makoto Hashimoto, Hikaru Kato, Ayako Katsuki, Sachiko Tsukamoto, Isao Fujii
Himeic acid A, produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin-activating enzyme E1 in the ubiquitin-proteasome system. To elucidate the mechanism of himeic acid biosynthesis, we first carried out feeding experiments with labeled precursors. The long fatty acyl side chain attached to the pyrone ring was confirmed to be of polyketide origin, while the amide substituent was found to be derived from leucine. These results suggest that a polyketide synthase-non-ribosomal peptide synthase (PKS-NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS-NRPS gene by Agrobacterium-mediated transformation confirmed that HimA PKS-NRPS is involved in himeic acid biosynthesis. We thus have identified the him biosynthetic gene cluster for himeic acid in A. japonicus MF275.
A Thiolactone Strategy for Straightforward Synthesis of Disulfide Linked Side Chain-to-tail Cyclic Peptides featuring a N-terminal modification handle ChemBioChem (IF 2.847) Pub Date : 2018-01-04 Dorien Van Lysebetten, Stefania Felissati, Eirini Antonatou, Lieselot Carrette, Pieter Espeel, Evelien Foquet, Filip Du Prez, Annemieke Madder
The development of straightforward and versatile peptide cyclisation methods is highly desired in order to meet the demand for more stable peptide based drugs. In this paper, a new method for the synthesis of side chain-to-tail cyclic peptides with simultaneous introduction of a N-terminal handle based on the introduction of a N-terminal thiolactone building block, is described. A primary amine liberates a homocysteine analogue from the thiolactone building block which further enables cyclisation of the peptide through disulfide bond formation with a C-terminal cysteamine. Post-cyclisation modification can be achieved by using small bifunctional amines. Alternatively, synthesis of lipopeptides is demonstrated through direct thiolacton opening with long-chain alkyl amines.
Design of aminobenzothiazole inhibitors of Rho Kinases 1 and 2 utilizing PKA as a structure surrogate ChemBioChem (IF 2.847) Pub Date : 2018-01-04 Russell Judge, Victoria Scott, Gricelda Simler, Steve Pratt, Marian Namovic, Brent Putman, Ana Aguirre, Vincent Stoll, Mulugeta Mamo, Steve Swann, Adrian D. Hobson
We describe the design, synthesis and SAR of a series of 2-aminobenzothiazole inhibitors of Rho Kinase (ROCK) 1 and 2 which were optimized to low nanomolar potencies using structure-based design. Protein Kinase A was used as a surrogate protein structure to guide compound design. A subset of these molecules also showed robust activity in a myosin phosphatase cell based assay and in a mechanical hyperalgesia in vivo pain model.
Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis ChemBioChem (IF 2.847) Pub Date : 2018-01-04 Ge Qu, Richard Lonsdale, Peiyuan Yao, Guangyue Li, Beibei Liu, Manfred T. Reetz, Zhoutong Sun
Alkoxy-tetrazine substitution at a boron center: a strategy for synthesizing highly fluorogenic hydrophilic probes ChemBioChem (IF 2.847) Pub Date : 2018-01-03 Min Wu, Xiaoai Wu, Yayue Wang, Lei Gu, Jiao You, Haoxing Wu, Ping Feng
Strongly fluorogenic BODIPY-tetrazine probes have been obtained by introducing an alkoxy-tetrazine fragment at the boron center. The fluorescence signal from these probes strongly increases by up to 225-fold after reaction with bioorthogonal coupling partners, and the hydrophilicity of probes is improved which is suitable for live-cell imaging.
Altering 2-hydroxybiphenyl 3-monooxygenase regioselectivity by protein engineering for the production of a new antioxidant ChemBioChem (IF 2.847) Pub Date : 2018-01-03 Almog Bregman-Cohen, Batel Deri, Shiran Maimon, Yael Pazy, Ayelet Fishman
2-Hydroxybiphenyl 3-monooxygenase (HbpA) is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing for the first time 3-hydroxybiphenyl, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity as the well-studied piceatannol. The crystal structure of M321A was determined (2.78 Å) and molecular docking of the 3-substituted phenol provided a rational explanation for the altered regioselectivity. Furthermore, HbpA was found to possess pro-S enantioselectivity towards the production of several chiral sulfoxides, while variant M321F exhibited improved enantioselectivity. Based on biochemical characterization of several mutants, it was suggested that Trp97 stabilizes the substrate in the active site, Met223 is involved in NADH entrance or binding to the active site, while Pro320 may facilitate FAD movement.
Cytochrome C catalyzes the hydrogen peroxide-assisted oxidative desulfuration of 2-thiouridines in transfer RNAs ChemBioChem (IF 2.847) Pub Date : 2017-12-29 Małgorzata Sierant, Katarzyna Kulik, Elzbieta Sochacka, Rafal Szewczyk, Milena Sobczak, Barbara Nawrot
The 5-substituted 2-thiouridines (R5S2U) present in the first (wobble) position of the anticodons of transfer RNAs (tRNAs) contribute to accuracy in reading mRNA codons and tune protein synthesis. Previously, we have shown that, under oxidative stress conditions in vitro, R5S2Us are sensitive to hydrogen peroxide (H2O2), and their oxidative desulfuration produces 5-substituted uridines (R5Us) and 4-pyrimidinone nucleosides (R5H2Us) at a ratio that depends on pH and an R5 substituent. Here, we demonstrate that the desulfuration of 2-thiouridines, either alone or within an RNA/tRNA chain, is catalyzed by cytochrome C (cyt C). Its kinetics is similar to this of Fenton-type catalytic S2U desulfuration. Cyt C/H2O2 and FeII-mediated reactions deliver predominantly H2U-type products. The pathway of the cyt C/H2O2 - peroxidase - mediated S2UH2U transformation through uridine sulfenic (U-SOH), sulfinic (U-SO2H) and sulfonic (U-SO3H) intermediates has been confirmed by LC-MS. The cyt C / H2O2 - mediated oxidative damage of S2U-tRNA may have biological relevance through the alteration of the cellular functions of transfer RNA.
Rational Design, Binding Studies and Crystal Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14-3-3ζ Adapter Protein ChemBioChem (IF 2.847) Pub Date : 2017-12-28 Carsten Schmuck, Martin Ehlers, Jean-Noel Grad, Sumit Mittal, David Bier, Marcel Mertel, Ludwig Ohl, Maria Bartel, Jeroen Briels, Marius Heimann, Christian Ottmann, Elsa Sanchez-Garcia, Daniel Hoffmann
14-3-3 proteins play a central role in signaling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14-3-3 proteins plays a key role in their activity. Here we present, to the best of our knowledge, the first example of a small molecule binding to the 14-3-3ζ dimerization interface. This compound was designed by rational in silico optimization of a peptidic ligand identified from a biochemical screening of a peptidic library and the binding was characterized by experimental binding studies, multiscale simulations and X-ray crystallography.
Stabilization of the Reductase Domain in the Catalytically Self-Sufficient Cytochrome P450BM3 via Consensus-Guided Mutagenesis ChemBioChem (IF 2.847) Pub Date : 2017-12-24 GLORIA SAAB-RINCON, Hanan Alwaseem, Valeria Guzmán-Luna, Leticia Olvera, Rudi Fasan
The multi-domain, catalytically self-sufficient cytochrome P450 BM-3 from Bacillus megaterium (P450BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus-guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3. Upon phylogenetic analysis of a set of distantly related P450s (% identity > 38%), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effect relative to the wild-type reductase domain. Recombination of the six most stabilizing mutations resulted in the generation of two thermostable variants featuring up to 10-fold longer half-lives at 50°C and increased catalytic performance at elevated temperature. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increase the thermal stability of the FAD-binding domain and that the optimal temperature (Topt) of the enzyme has shifted from 25°C to 40°C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multi-domain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.
Evaluation of β-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure ChemBioChem (IF 2.847) Pub Date : 2017-12-22 David E Mortenson, Dale F Kreitler, Nicole C Thomas, Ilia A Guzei, Samuel H Gellman, Katrina T. Forest
β-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and β residues have been widely investigated as subunits in protein-like molecules that adopt discrete and predictable conformations. Two classes of β residue have been widely explored in the context of generating α-helix-like conformations: β3-amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen; and residues constrained by a five-membered ring, such as the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their β3 homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained β residue, however, can offset the destabilizing effect of α➔β substitution. Here we extend the study of α➔β substitutions, involving both β3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a 2-, 3- or 4-residue β-hairpin loop, and we evaluate α➔β replacements at each loop position for each variant. By referral to the φ,ψ angles of the native structure, a stereochemically appropriate ACPC residue can be chosen. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three β-containing Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.
ChemBioChem–All Areas of Chemistry and Biology Intertwined ChemBioChem (IF 2.847) Pub Date : 2017-12-21 Ruben Ragg
Characterization of CYP154F1 from Thermobifida fusca YX and extension of its substrate spectrum by site-directed mutagenesis ChemBioChem (IF 2.847) Pub Date : 2017-12-19 Ansgar Rühlmann, Georg Groth, Vlada B. Urlacher
Previous studies on cytochrome P450 monooxygenases (CYP) from the family 154 reported on their substrate promiscuity and high activity. Hence, in this study we describe the uncharacterized family member CYP154F1. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8-10 atoms. In contrast to the thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and low activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids resulted in up to 10-fold higher conversion rates of small substrates such as geraniol. Replacement of T234 by small hydrophobic amino acids valine or alanine resulted in mutants with extended substrate spectra. They were able to convert some of the larger substrates of CYP154E1 such as (E)-stilbene and (+)-nootkatone.
Heat shock proteins revisited using a mutasynthetically generated reblastatin library for comparing the inhibition of human with Leishmania Hsp90s ChemBioChem (IF 2.847) Pub Date : 2017-12-18 Andreas Kirschning, Jekaterina Ongouta, Simone Eichner, Klaus Kock, Frank Stahl, Florenz Sasse, Júlio César Borges, Fernanda Aparecida Heleno Batista, Christian Hermann, Sona Mohammadi-Ostad-Kalayeh, Thomas Scheper, Carsten Zeilinger
Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo-biosynthetic approach using an AHBA(-) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potency of these mutaproducts and an extended library of natural products and derivatives was probed with purified Hsps, obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activity of potential inhibitors using a displacement assay in which fluorescent-labelled ATP competes for the ATP binding sites of Hsps in the presence of the respective inhibitor. The results were compared with cell-based assays and in selected cases with calorimetry ITC measurements. In essence, reblastatin derivatives are also able to effectively bind to the ATP-binding site of LbHsp90 and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets.
Biochemical characterization and mechanistic analysis of the levoglucosan kinase from Lipomyces starkeyi ChemBioChem (IF 2.847) Pub Date : 2017-12-18 Christina Rother, Alexander Gutmann, Ramakrishna Gudiminchi, Hansjörg Weber, Alexander Lepak, Bernd Nidetzky
Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-D-glucopyranose) in the presence of Mg2+-ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10°C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-D-glucose-6-phosphate, in a manner reminiscent of an inverting β-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20-50 mM), the apparent binding of levoglucosan (Km = 180 mM) and ATP (Km = 1.0 mM) as well as the inhibition by ADP (Ki = 0.45 mM) and D-glucose-6-phosphate (IC50 = 56 mM). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side and no enzymatic back reaction from D-glucose-6-phosphate and ADP was observed in a broad range of conditions. 6-Phospho-α-D-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol (6-phospho-D-glucal) were synthesized as probes of the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring-flip 4C1 1C4 required for 1,6-anhydro-product synthesis from D-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.
Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting ChemBioChem (IF 2.847) Pub Date : 2017-12-18 Kathrin Lang
O⁴-Alkylated-2'-deoxyuridine repair by O⁶-alkylguanine DNA alkyltransferase is augmented by a C5-fluorine modification ChemBioChem (IF 2.847) Pub Date : 2017-12-15 Lauralicia Sacre, Derek K. O'Flaherty, Philippe Archambault, William Copp, Gilles Peslherbe, Heidi M. Muchall, Christopher James Wilds
Oligonucleotides containing various adducts including ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl groups at the O⁴-atom of 5-fluoro-O⁴-alkyl-2′-deoxyuridine were prepared by solid-phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilized the duplex by approximately 10 ºC relative to the control containing 5-fluoro-2′-deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B-form DNA structure. O⁶-alkylguanine DNA alkyltransferase (AGT) from human (hAGT) was most efficient at repair of the 5-fluoro-O⁴-benzyl-2′-deoxyuridine adduct whereas the thymidine analog was refractory to repair. The E. coli AGT variant (OGT) was also efficient at removing O⁴-ethyl and benzyl adducts of 5-fluoro-2-deoxyuridine. Computational assessment of N1-methyl analogs of the O⁴-alkylated nucleobases revealed that the C5-fluorine modification had an influence of reducing the electron density of the O⁴-Cα bond relative to thymine (C5-methyl) and uracil (C5-hydrogen). These results reveal the positive influence of the C5-fluorine atom on repair of larger O⁴-alkyl adducts expanding our knowledge of the range of substrates that are able to be repaired by AGT.
NMR study of Rcf2 reveals an unusual dimeric topology in detergent micelles ChemBioChem (IF 2.847) Pub Date : 2017-12-14 Shu Zhou, Pontus Pettersson, Peter Brzezinski, Pia Ädelroth, Lena Mäler
The Saccharomyces (S.) cerevisiae mitochondrial respiratory supercomplex factor 2 (Rcf2) plays a role in assembly of supercomplexes composed of cytochrome bc1 (complex III) and cytochrome c oxidase (complex IV). The Rcf2 protein was here expressed in E. coli, refolded and reconstituted into dodecylphosphocholine (DPC) micelles. The structural properties of Rcf2 were studied by solution NMR and near complete backbone assignment of Rcf2 was achieved. The secondary structure of Rcf2 contains seven helices, of which five are putative transmembrane (TM) helices, including, unexpectedly, a region formed by a charged 20-residue helix at the C-terminus. Further studies demonstrated that Rcf2 forms a dimer and the charged TM helix is involved in this dimer formation. Our results provide a basis for understanding the role of this assembly/regulatory factor in supercomplex formation and function
Novel 4-amino-1,2,3-triazole inhibitors of indoleamine 2,3-dioxygenase form a long-lived complex with the enzyme and display exquisite cellular potency ChemBioChem (IF 2.847) Pub Date : 2017-12-14 Julie Alexandre, Michael Swan, Mike Latchem, Dean Boyall, John Pollard, Stuart Hughes, James Westcott
Indoleamine-2,3 dioxygenase (IDO) has emerged as a central regulator of immune responses in both normal and disease biology. Due to its established role in promoting tumour immune escape, IDO has become an attractive target for cancer treatment. We have identified a novel series of highly cell potent IDO inhibitors based on a 4-amino-1,2,3-triazole core. Comprehensive kinetic, biochemical and structural studies demonstrate that compounds from this series have a non-competitive kinetic mechanism-of-action with respect to the tryptophan substrate. Surprisingly, they bind in the tryptophan pocket, making a direct ligand interaction with the haem iron of the porphyrin cofactor. We propose that these data can be rationalised by an ordered binding mechanism in which the inhibitor binds an apo form of the enzyme that is not competent to bind tryptophan. These inhibitors also form a very tight, long-lived complex with the enzyme, which we propose partially explains their exquisite cellular potency. This novel series represents an attractive starting point for the future development of potent IDO targeted drugs.
DNA synthesis by primer exchange reaction cascades ChemBioChem (IF 2.847) Pub Date : 2017-12-14 Marcel Hollenstein
The autonomous synthesis of single-stranded DNA molecules of arbitrary size and sequence composition can easily be achieved by Primer Exchange Reaction (PER) cascades. In PER cascades, the sequential polymerase-mediated extension of DNA primers is guided by catalytic hairpins. This highlight presents this method and illustrates its potential for applications in DNA nanotechnology.
Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR ChemBioChem (IF 2.847) Pub Date : 2017-12-13 John M. Egner, Davin R. Jensen, Mike D. Olp, Nolan W. Kennedy, Brian F. Volkman, Francis C. Peterson, Brian C. Smith, R Blake Hill
An academic chemical screening approach was developed and a 352-chemical fragment library screened against 3 different protein targets using 2D protein-detected NMR. The approach was optimized against 2 protein targets with known ligands, CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced NMR crosspeak broadening, but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric - difference intensity analysis (DIA) - was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened and 6 hits were identified using DIA. Screening these diverse protein targets identified quinones and catechols that induced NMR crosspeak broadening, hampered NMR analyses, but are currently not computationally identified as pan-assay interference compounds. The results establish a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3 - 1.6 mM. DIA may prove useful in library screening and other applications where NMR chemical shift perturbations are measured.
Flow-Alignment of Extracellular Vesicles: Structure and Orientation of Membrane Associated Biomacromolecules Studied with Polarized Light ChemBioChem (IF 2.847) Pub Date : 2017-12-13 Imola Cs. Szigyarto, Róbert Deák, Judith Mihály, Sandra Rocha, Ferenc Zsila, Zoltán Varga, Tamás Beke-Somfai
Extracellular vesicles (EVs) are currently in the scientific focus having a great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, thus additional insight to molecular level properties, membrane-protein interactions, or membrane rigidity is still needed. We here demonstrate using red blood cell derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular level structural information on these systems. Flow-linear dichroism (flow-LD) measurements show that EVs can be oriented by shear force, and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage this interaction ceases coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight to vesicle rigidity, which decreases in time parallel to changes in protein conformation. In overall, we propose that both LD and circular dichroism (CD) spectra provide simple, rapid, yet efficient ways to track changes in membrane-protein interactions of EV components at the molecular level which may also give insight to processes occurring during vesiculation.
Not oligomers but amyloids are cytotoxic in the membrane-mediated amyloidogenesis by amyloid-β peptide ChemBioChem (IF 2.847) Pub Date : 2017-12-13 Naoya Itoh, Eri Takada, Kaori Okubo, Yoshiaki Yano, Masaru Hoshino, Akira Sasaki, Masataka Kinjo, Katsumi Matsuzaki
The formation of neurotoxic aggregates by amyloid-β peptide (Aβ) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous-phase aggregation of Aβ. Membrane-mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Here, the aggregational processes of Aβ on living cells and cytotoxic events were monitored by fluorescence techniques. Aβ formed amyloids after forming oligomers composed of ~10 Aβ molecules. The formation of amyloids was necessary to activate apoptotic caspase-3 and reduce the cell proliferation ability, indicating that amyloid formation is a key event in Aβ-induced cytotoxicity.
Peptide-Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real-Time Detection of Lysine Deacylation ChemBioChem (IF 2.847) Pub Date : 2017-12-12 Debra R Rooker, Yuliya Klyubka, Ritika Gautam, Elisa Tomat, Daniela Buccella
Histone deacetylases regulate acetylation levels of numerous proteins and play key roles in physiological processes as well as disease states. In addition to acetyl groups, deacetylases can remove other acyl modifications on lysines whose roles and regulation are far less understood. We report herein a peptide-based fluorescent probe for single-reagent, real time detection of deacetylase activity that can be readily adapted for probing broader lysine deacylation, including decrotonylation. Following cleavage of the lysine modification, the probe undergoes rapid intramolecular imine formation that results in marked optical changes, thus enabling convenient detection of deacylase activity with good statistical Z' factors for both absorption and fluorescence modalities. The peptide-based design offers broader isozyme scope than small molecule analogues, and is suitable for probing both metal- and NAD+-dependent deacetylases. With an effective sirtuin activity assay in hand, we demonstrate that iron chelation by Sirtinol, a commonly employed sirtuin inhibitor, results in an enhancement in the inhibitory activity of the compound that may affect its performance in vivo.
Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications ChemBioChem (IF 2.847) Pub Date : 2017-12-13 Veeresh Juturu, Jin Chuan Wu
Evolving the N-Terminal Domain of Pyrrolysyl-tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids ChemBioChem (IF 2.847) Pub Date : 2017-12-13 Vangmayee Sharma, Yu Zeng, W. Wesley Wang, Yuchen Qiao, Yadagiri Kurra, Wenshe R. Liu
Epitope Resolved Detection of Peanut Specific IgE Antibodies by SPR Imaging ChemBioChem (IF 2.847) Pub Date : 2017-12-12 Min Shen, Amit Joshi, Raghu Vannam, Chandra Dixit, Robert Hamilton, Challa V Kumar, James F Rusling, Mark Wayne Peczuh
Peanut allergy can be life-threatening and is mediated by allergen-specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes identifies specific interactions underlying the allergic response. Here we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope resolved detection. IgE antibodies are first captured by magnetic beads bearing IgE ε-chain specific antibodies and then introduced to an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses are achieved by establishing a binding environment that minimizes cross-reactivity while maximizes analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from 10 μL patient serum samples in a 45 min assay. Excellent correlation of Ara h2-specific IgE values was found between ImmunoCAP assays and the new SPRi method.
Antibiotic Algae by Chemical Surface Engineering ChemBioChem (IF 2.847) Pub Date : 2017-12-12 Isabel Kerschgens, Karl Gademann
Chemical cell surface engineering is a valuable tool for modifying and altering cellular functions. Herein we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4-hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability and motility of the cells. The modified cells were used to inhibit bacterial growth of Gram-positive Bacillus subtilis cultures. Delivery of the antibiotic form the microalgae to the bacterial cells is supported by microscopy. Our studies provide compelling evidence that (a) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and that (b) living microalgae can serve as new platforms for drug delivery.
Synthetic cancer-targeting innate immune stimulators give insights into avidity effects ChemBioChem (IF 2.847) Pub Date : 2017-12-12 Anne Claire Conibear, André Pötgens, Karine Thewes, Claudia Altdorf, Clarissa Hilzendeger, Christian Friedrich Wilhelm Becker
Multispecific and multivalent antibodies are seen as promising cancer therapeutics and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study, we synthesized a series of targeted immune system engagers (ISErs) using solid phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two 'binder' peptides that target ephrin A2 and integrin α3 receptors and an 'effector' peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anti-cancer therapeutics.
Flavin-N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme ChemBioChem (IF 2.847) Pub Date : 2017-12-12 Yumin Dai, Karina Kizjakina, Ashley C. Campbell, David A. Korasick, John J. Tanner, Pablo Sobrado
Ligand-Induced Dimerization of a Truncated Parallel MYC G-Quadruplex ChemBioChem (IF 2.847) Pub Date : 2017-12-11 Andrea Funke, Beatrice Karg, Jonathan Dickerhoff, Darko Balke, Sabine Müller, Klaus Weisz
Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry. The targeted MYC3 sequence lacks 3'-flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt-ended 3'-outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm a ligand-induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3'-outer tetrads. Additional ligand molecules in excess bind to the 5'-outer tetrads of the sandwich complex. Such a ligand-promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex-based technologies.
Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism ChemBioChem (IF 2.847) Pub Date : 2017-12-11 Jennifer I. Brown, Timo Koopmans, Jolinde van Strien, Nathaniel I. Martin, Adam Frankel
The cover feature picture shows the cofactor SAM (teal) and the peptide binding groove (pink) within a PRMT1 dimer. We find that PRMT1 binds its substrates by using a sequential ordered mechanism in which binding of the cofactor SAM precedes binding of the target peptide similar to how putting on socks precedes putting on shoes—a handy metaphor. More information can be found in the full paper by N. I. Martin, A. Frankel et al.
Characterisation of CYP102A25 from B. marmarensis and CYP102A26 from P. halophilus: P450 homologues of BM3 with preference towards hydroxylation of medium chain fatty acids ChemBioChem (IF 2.847) Pub Date : 2017-12-08 Joanne L Porter, Jack Manning, Selina Sabatini, Michele Tavanti, Nicholas J Turner, Sabine L Flitsch
Cytochrome P450 monooxgenases are highly desired biocatalysts due to their ability to catalyse a wide variety of chemically challenging C-H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self-sufficient proteins consisting of a haem and FMN-FAD reductase domain fused in a single component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω-1, ω-2 and ω-3 hydroxy acids. These monooxygenases have broad applications in biotechnology however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein we present the characterization of two new members of this class of enzymes: CYP102A25 (BMar) from B. marmarensis and CYP102A26 (PHal) from P. halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid chain saturated (C14-18) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for hydroxylation of myristic acid targeting the ω-2 position for C-H activation.
The Anthelmintic Drug Niclosamide and its Analogues Activate the Parkinson's Disease Associated Protein Kinase PINK1 ChemBioChem (IF 2.847) Pub Date : 2017-12-10 Erica Barini, Ageo Miccoli, Federico Tinarelli, Katie Mulholand, Hachemi Kadri, Farhat Khanim, Laste Stojanovski, Kevin D Read, Kerry Burness, Julian J Blow, Youcef Mehellou, Miratul Muqit
Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases such as PD. Herein, we show that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells via reversible impairment of the mitochondrial membrane potential. Using these compounds, we demonstrate for the first time that the PINK1 pathway is active and detectable in primary neurons. Our findings suggest that niclosamide and its analogues are robust compounds to study the PINK1 pathway and may hold promise as a therapeutic strategy in Parkinson's and related disorders.
A single mutation increases the activity and stability of Pectobacterium carotovorum nitrile reductase ChemBioChem (IF 2.847) Pub Date : 2017-12-07 Zheng Zhou, Min Li, Jian-He Xu, Zhi-Jun Zhang
Nitrile reductase is considered as a promising and environmentally benign nitrile-reducing biocatalyst to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of nitrile reductases reported so far are very low. To date, any attempts by protein engineering to increase the catalytic activity of nitrile reductase have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 U·gprot-1 to 526 U·gprot-1 by engineering the substrate binding pocket, meanwhile the thermostability was also improved (ca. 2 folds), showing half-lives of 140 h and 32 h at 30 ºC and 40 ºC, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1), the variant was advantageous over the wild-type enzyme with higher reaction rate and complete conversion of the substrate with a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductase to further increase the catalytic efficiency, which is a prerequisite for applying this novel biocatalyst in synthetic chemistry.
MicroRNA Detection by DNA-Mediated Liposome Fusion ChemBioChem (IF 2.847) Pub Date : 2017-12-07 Coline Jumeaux, Olov Wahlsten, Stephan Block, Eunjung Kim, Philip D Howes, Rona Chandrawati, Fredrik Höök, Molly Stevens
Membrane fusion is a process of fundamental importance in biological systems, involving highly selective recognition mechanisms for trafficking of molecular and ionic cargos. Mimicking natural membrane fusion mechanisms for the purpose of biosensor development holds great potential for amplified detection, since relatively few highly discriminating targets lead to fusion and an accompanied engagement of a large payload of signal-generating molecules. In this work, we use sequence specific DNA-mediated liposome fusion for highly selective detection of microRNA. We demonstrate detection of miR-29a, a known flu biomarker, down to 18 nM within 30 minutes with high specificity using a standard laboratory microplate reader. Further, we demonstrate one order of magnitude improvement in the limit of detection by using a novel imaging technique combined with an intensity fluctuation analysis, coined 2-color fluorescence correlation microscopy.
Euphorbia tirucalli β-amyrin cyclase: Trp612 and Tyr736 π-electrons participate in a cation-π interaction with the transient cation and a CH-π interaction with Leu734, respectively ChemBioChem (IF 2.847) Pub Date : 2017-12-06 Yukari Aiba, Takumi Watanabe, Yuri Terasawa, Chiaki Nakano, Tsutomu Hoshino
The functions of Trp612, Leu734 and Tyr736 of Euphorbia tirucalli β-amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5% of the wild-type activity for the W612H variant, 43% for W612F, and 63% for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π-electron density. Thus, the major function of Trp612 is to stabilize transient cations via a cation-π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E-ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile and Met variants showed markedly high activities (56─78% of wild-type activity); therefore, the appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67%), indicating that the π-electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and situated face to face, suggesting that a CH-π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates via a cation-π interaction.
A T7 RNA Polymerase Mutant Enhances the Yield of 5′-Thienoguanosine-Initiated RNAs ChemBioChem (IF 2.847) Pub Date : 2017-12-07 Seth Lyon, Venkat Gopalan
Spontaneous covalent self-assembly of the Azoarcus ribozyme from five fragments ChemBioChem (IF 2.847) Pub Date : 2017-12-05 Tharuka S. Jayathilaka, Niles Lehman
Spontaneous covalent assembly of short RNA fragments has been proposed as a plausible prebiotically relevant pathway to a self-reproducing system. We had previously shown that the Azoarcus group I intron could self-assemble from four RNA fragments. Here, we extend this fragmentation to five RNAs that average <40 nucleotides in length. We optimize this reaction and show that a dehydration-rehydration sequence is the most effective means to date to shift the self-assembly equilibrium from reactants to products.
Alphanumerical Visual Display Made of DNA Logic Gates for Drug Susceptibility Testing of Pathogens ChemBioChem (IF 2.847) Pub Date : 2017-12-05 Ryan Connelly, Evgeny S Morozkin, Yulia Gerasimova
Molecular diagnostics of drug resistant pathogens requires analysis of point mutations in bacterial or viral genomes, which is usually performed by trained professionals and/or by a sophisticated computer algorithm. Here, we developed a DNA-based logic system that autonomously analyzes mutations in the genome of Mycobacterium tuberculosis complex (MTC) bacterial species and communicates the output to a human user as alphanumeric characters read by the naked eye. The five-gate system displays "O" ("no infection") for the absence of MTC infection; and "P" or "F" for passing or failing a drug susceptibility test, respectively.
A Chemoenzymatic Histology Method for O-GlcNAc Detection ChemBioChem (IF 2.847) Pub Date : 2017-12-05 Aime Lopez Aguilar, Xiaomeng Hou, Liuqing Wen, Peng G. Wang, Peng Wu
Specificity of Donor Structures for endo-β-N-Acetylglucosaminidase-Catalyzed Transglycosylation Reactions ChemBioChem (IF 2.847) Pub Date : 2017-12-04 Nozomi Ishii, Ken Ogiwara, Kanae Sano, Jyunichi Kumada, Kenji Yamamoto, Yuji Matsuzaki, Ichiro Matsuo
Characterization of Terminators in Saccharomyces cerevisiae and an Exploration of Factors Affecting Their Strength ChemBioChem (IF 2.847) Pub Date : 2017-12-04 Linna Wei, Zhaoxia Wang, Genlin Zhang, Bangce Ye
The effects of pressure (0.1 - 200 MPa) and pH on the hydrolysis of cytosine at 373 K: implications for nucleotide stability around deep-sea black smokers ChemBioChem (IF 2.847) Pub Date : 2017-12-03 Christopher P Lepper, Martin AK Williams, David Penny, Patrick JB Edwards, Geoffrey Brind Jameson
The relatively low chemical stability of cytosine compared to other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil has remained unknown. We have determined by in situ NMR measurements that the half-life of cytosine at 373.15 K decreases from 18.0 (±0.7) days at ambient pressure (0.1 MPa) to 8.64 (±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of -11.8 (±0.5) cm3 mol-1, a decrease that is similar to the molar volume of water (18.0 cm3 mol-1) and consistent with a tetrahedral 3,3-hydroxyamine transition-state/intermediate species. Very similar behavior was observed also for cytidine. At both ambient and high pressures, the half-life of cytosine decreases significantly as pH is dropped from 7.0 to 6.0. These results provide scant support for the notion that RNA-based life forms originated in high-temperature, high-pressure, acidic environments.
Protein Folding by Water-Soluble Cyclic Diselenides with Novel Oxidoreductase and Isomerase Activities ChemBioChem (IF 2.847) Pub Date : 2017-12-02 Kenta Arai, Haruhito Ueno, Yuki Asano, Gaurango Chakrabarty, Shingo Shimodaira, Govindasamy Mugesh, Michio Iwaoka
Protein disulfide isomerase (PDI) family, present in the endoplasmic reticulum (ER) of mammalian cells, catalyzes the formation and cleavage of disulfide bonds and thereby helps in protein folding. A decrease in PDI activity under ER stress conditions lead to protein misfolding, which is responsible for the progression of various human diseases, such as Alzheimers, Parkinsons, diabetes mellitus and atherosclerosis. In this paper, we report that water-soluble cyclic diselenides mimic the multifunctional activity of PDI family by facilitating oxidative folding, disulfide formation/reduction and repair of the scrambled disulfide bonds in misfolded proteins.
Unravelling the carbohydrate-binding preferences of the carbohydrate-binding modules of AMP-activated protein kinase. ChemBioChem (IF 2.847) Pub Date : 2017-11-30 Jesse Mobbs, Alex di Paolo, Riley Metcalfe, Emily Selig, David Stapleton, Michael Griffin, Paul Gooley
The β-subunit of AMP-activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a Carbohydrate Binding Module (CBM) that interacts with glycogen. While the β1- and β2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. β2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and we have recently shown that a Thr insertion in β2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2-CBM binds all carbohydrate with stronger affinity. Here we show that residue 134 (Val for β2 and Thr for β1), which does not contact carbohydrate, appears to account for the affinity difference. Characterization by NMR, however, suggests that the mutant β2-Thr101Δ/Val134Thr differs from β1-CBM, and the mutant β1- Thr101ins/Thr134Val differs from β2-CBM. Furthermore, these mutants were less stable to chemical denaturation compared to wild type β-CBMs confounding the affinity analyses. To support the importance of Thr101 and Val134 we constructed the ancestral CBM. This CBM retained Thr101 and Val134 suggesting that the extant β1-CBM has a modest loss of function in carbohydrate binding. As the ancestor bound carbohydrate with equal affinity to β2-CBM we conclude that residue 134 does play an indirect role in carbohydrate binding.
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