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Chembiochem: a European Journal of Chemical Biology

Carsten Schmuck, Sandra Junghänel, Sarah Karczewski, Sandra Bäcker, Shirley Knauer
We discovered a new class of artificial peptidic transfection vectors based on an artificial anion binding motif, the guanidiniocarbonylpyrrole cation (abbreviated as GCP). This new type of vector is surprisingly small compared to traditional systems and our previous work suggested that the GCP group is important for promoting the critical endosomal escape. We now present here a systematic comparison of similar DNA-ligands featuring our GCP oxo-anion binding motif with DNA-ligands only consisting of natural occurring amino acids...
September 15, 2017: Chembiochem: a European Journal of Chemical Biology
Leonhard Kick, Sabrina Harteis, Maximilian F Koch, Sabine Schneider
Inteins carry out protein-splicing reactions, which are utilised in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in a head-to-tail cyclisation of the target sequence, which can be used to genetically encode and express libraries of cyclic peptides. The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess the cyclic peptide formation of different, hexameric target sequences in vivo by the DnaE split-inteins from Synechocystis sp...
September 15, 2017: Chembiochem: a European Journal of Chemical Biology
Takashi Jin, Setsuko Tsuboi
Deregulation in apoptosis induces numerous diseases such as cancer, cardiovascular and neurodegenerative diseases. The detection of apoptotic cells is crucial for understanding the mechanism of those diseases and for therapy development. Although optical imaging using visible-emitting fluorescent probes such as FITC-labeled annexin V is widely used for the detection of apoptotic cells, there are a very limited probes that can be used in near-infrared (NIR) region over 700 nm. Compared with visible light, NIR light is highly permeable in turbid biological samples and tissues...
September 13, 2017: Chembiochem: a European Journal of Chemical Biology
Shankar Balasubramanian, Arun Ankush Tanpure
The synthesis of 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm), 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C) phosphoramidite monomers has been developed. Optimization of mild post-synthetic deprotection conditions enabled the synthesis of RNA containing all four naturally occurring cytosine-modifications (hm5Cm, hm5C, f5C plus 5-methylcytosine). Given the considerable interest in RNA modifications and epitranscriptomics, the availability of synthetic monomers and RNAs containing these modifications will be valuable to elucidate their biological function(s)...
September 13, 2017: Chembiochem: a European Journal of Chemical Biology
Lisa C Kühnel, Bettina M Nestl, Bernhard Hauer
Squalene hopene cyclases (SHC) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Furthermore, the addition of small chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and thus, to a significant alteration of the product pattern compared to wildtype...
September 12, 2017: Chembiochem: a European Journal of Chemical Biology
Jeroen Sidney Dickschat, Jan Rinkel, Tim Klapschinski, Jörn Petersen
The L-cystine-b-lyase from Phaeobacter inhibens is involved in the biosynthesis of the sulfur containing antibiotic tropodithiethic acid. The recombinant enzyme was obtained by heterologous expression in Escherichia coli and biochemically characterised by unambiguous chemical identification of the products formed from the substrate L-cystine, investigation of the substrate spectrum, determination of the enzyme kinetics, sequence alignment with closely related homologs and site-directed mutagenesis to identify a highly conserved lysine residue that is critical for functionality...
September 12, 2017: Chembiochem: a European Journal of Chemical Biology
Sureshbabu Nagarajan, Lisa Jill Lapidus
The aggregation of intrinsically disordered and misfolded proteins in the form of oligomers and fibrils plays a crucial role in a number of neuro and non-neurodegenerative diseases. Currently, most probes and biophysical techniques that detect and characterize fibrils to high resolution fail to show sensitivity and binding for oligomers. Here we show 9-(dicyano-vinyl) julolidine (DCVJ), a class of molecular rotor, binds Aβ early aggregates and reports on the kinetics as well as packing of oligomer formation...
September 11, 2017: Chembiochem: a European Journal of Chemical Biology
Md Kausar Alam, Carolina Gonzalez, Wayne Hill, Humphrey Fonge, Kris Barreto, Clarence Ronald Geyer
Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive as each new recombinant antibody needs to be optimized for expression, stability, bio-distribution, and pharmacokinetics. We developed a new way to construct recombinant antibody-like "devices" using a "bottom up" approach to build them from well-behaved discrete recombinant antibody domains or "parts". Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation...
September 10, 2017: Chembiochem: a European Journal of Chemical Biology
Dana Colpa, Nikola Loncar, Mareike Schmidt, Marco Fraaije
In this work we prepared a set of bifunctional oxidase-peroxidases by fusing four distinct oxidases to a peroxidase. While such fusion enzymes have not been observed in nature, they could be expressed and purified with good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. The peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for selective detection of xylitol and cellobiose with a detection limit in the low µM range...
September 8, 2017: Chembiochem: a European Journal of Chemical Biology
Bharathikumar Vellalore Maruthachalam, Ayman El-Sayad, Jianghai Liu, Wayne Hill, Ashley Sutherland, Landon Pastushok, Humphrey Fonge, Kris Barreto, Clarence Ronald Geyer
Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage-displayed Fab library built on a modified trastuzumab framework with four fixed and two diversified complementarity-determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most-frequent 'canonical' CDR conformation preferred by the modified trastuzumab Fab framework. The library diversity was engineered within CDRs L3 and H3 using custom-designed trinucleotide phosphoramidite mixes and biased towards human antibody CDR sequences...
September 7, 2017: Chembiochem: a European Journal of Chemical Biology
Elena Mutti, Miriam Hunger, Sergey Fedosov, Ebba Nexo, Bernhard Kräutler
We report on the synthesis and structural characterization of Co-(dN)25-Cbl and Co-(dN)39-Cbl, organometallic DNA-B12-conjugates with single DNA-strands consisting of 25 and 39 deoxy-nucleotides, respectively, and on binding studies of these two DNA-cobalamin (DNA-Cbl) conjugates to the three homologous human Cbl transporting proteins transcobalamin (TC), intrinsic factor (IF) and haptocorrin (HC). This investigation tests the suitability of such DNA-Cbls for the task of eventual in-vivo oligonucleotide delivery...
September 7, 2017: Chembiochem: a European Journal of Chemical Biology
Liudmila Tenkovskaia, Mizuki Murakami, Kotone Okuno, Daijiro Ueda, Tsutomu Sato
Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl-β-curcumene (C35 ) but a bicyclic scaffold from squalene (C30 ) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes...
September 7, 2017: Chembiochem: a European Journal of Chemical Biology
Mary C Andorfer, Ketaki D Belsare, Anna M Girlich, Jared Lewis
The remarkable site-selectivity and broad substrate scope of flavin dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site-selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required...
September 6, 2017: Chembiochem: a European Journal of Chemical Biology
Shun Nakano, Tomoki Tamura, Raj Kumar Das, Eiji Nakata, Young-Tae Chang, Takashi Morii
One of the key determinants to successfully obtain a useful biosensor is wide variation of fluorophores attached to a given receptor. Thus, use of a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. However, no effective method has yet been developed for constructing such a diverse library of fluorophore-modified receptors. Herein, we report a method for constructing fluorophore-modified receptors using a chemical library of synthetic fluorophores with a thiol-reactive group...
September 6, 2017: Chembiochem: a European Journal of Chemical Biology
Yuan Tian, Yanhong Jiang, Jingxu Li, Dongyuan Wang, Hui Zhao, Zigang Li
Stapled peptides emerged as a new class of targeting molecules of high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes were exceptionally intriguing to the field, yet elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on physiochemical properties and cell permeability of stapled α-helical peptides with different types of cross-links...
September 6, 2017: Chembiochem: a European Journal of Chemical Biology
Tsutomu Hoshino, Kazuya Nakagawa, Yukari Aiba, Daichi Itoh, Chika Nakada, Yukari Masukawa
The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli β-amyrin synthase were revealed by comparing the enzyme activities of the site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 dominantly afforded monocyclic camelliol C, suggesting that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation...
September 5, 2017: Chembiochem: a European Journal of Chemical Biology
Fumihiro Ishikawa, Shota Kasai, Hideaki Kakeya, Genzoh Tanabe
Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, serving as covalent tethers for amino acid and aryl acid substrates and enabling the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of aryl acid A-domains...
September 4, 2017: Chembiochem: a European Journal of Chemical Biology
Ikuro Abe, Takaaki Mitsuhashi, Masahiro Okada
We identified two unusual diterpene synthases composed of three domains (α, β, and γ) from fungal Penicillium species. They are the first enzymes found to possess both Type II terpene cyclase (TC) and prenyltransferase (PT) activities. These enzymes were characterized by heterologous expression in Aspergillus oryzae and in vitro experiments with wild-type, mutated, and truncated enzymes. The results revealed that the α domain in the C-terminal region of these enzymes was responsible for the PT activity, while the βγ domains in the N-terminal region composed the Type II TC, and formed copalyl diphosphate 2...
September 4, 2017: Chembiochem: a European Journal of Chemical Biology
Akimasa Miyanaga, Ryuichi Takayanagi, Takashi Furuya, Ayano Kawamata, Tomohiro Itagaki, Yoshiharu Iwabuchi, Naoki Kanoh, Fumitaka Kudo, Tadashi Eguchi
GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. The docking models, in conjunction with the results of the enzymatic assay with substrate analogues and site-directed mutagenesis suggested two distinct substrate binding modes for epoxidation and hydroxylation reactions, which explained how GfsF regulates the order of two oxidative reactions...
September 4, 2017: Chembiochem: a European Journal of Chemical Biology
Md Raihan Sarkar, Joel H Z Lee, Stephen Graham Bell
The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency compared to norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which in this enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was modified to a phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates...
September 3, 2017: Chembiochem: a European Journal of Chemical Biology
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