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Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis
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Joshua N. Kolev Jacqueline M. Zaengle Rajesh Ravikumar Prof. Rudi Fasan 《Chembiochem : a European journal of chemical biology》2014,15(7):1001-1010
The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active‐site positions of a substrate‐promiscuous CYP102A1 variant. The resulting “uP450s” were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small‐molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para‐acetyl‐Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp3)?H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity‐enhancing effect of active‐site substitutions involving the unnatural amino acid para‐amino‐Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts. 相似文献
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Annemieke de Jong Dr. Remco Merkx Dr. Ilana Berlin Dr. Boris Rodenko Ruud H. M. Wijdeven Dris El Atmioui Zeliha Yalçin Prof. Dr. Craig N. Robson Prof. Dr. Jacques J. Neefjes Dr. Huib Ovaa 《Chembiochem : a European journal of chemical biology》2012,13(15):2251-2258
Epitope‐tagged active‐site‐directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small‐molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor‐intensive intein‐based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active‐site‐directed probes and their application to activity‐based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull‐down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in‐gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull‐down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells. 相似文献
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Dagan Hadar Sigal Gelkop Livne Vaserman Dr. Miriam Amiram 《Chembiochem : a European journal of chemical biology》2021,22(8):1379-1384
Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment – typically at a single site or at a few sites per protein – of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l -phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production – using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain – of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling. 相似文献
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Dr. Juan Tang Chenfei Yu Axel Loredo Yuda Chen Prof. Dr. Han Xiao 《Chembiochem : a European journal of chemical biology》2021,22(3):501-504
Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins. 相似文献
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A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated. 相似文献
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Taehyung C. Lee Dr. Minjin Kang Dr. Chan Hyuk Kim Prof. Dr. Peter G. Schultz Prof. Dr. Eli Chapman Ashok A. Deniz 《Chembiochem : a European journal of chemical biology》2016,17(11):981-984
Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single‐molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site‐specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide–alkyne chemistry to attach an smFRET dye pair to site‐specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter‐ or intradomain structural features. An initial proof‐of‐principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines. 相似文献
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Development of Diubiquitin‐Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes
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Dr. Paul P. Geurink Bianca D. M. van Tol Duco van Dalen Paul J. G. Brundel Tycho E. T. Mevissen Dr. Jonathan N. Pruneda Dr. Paul R. Elliott Gabriëlle B. A. van Tilburg Dr. David Komander Prof. Dr. Huib Ovaa 《Chembiochem : a European journal of chemical biology》2016,17(9):816-820
Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′‐Boc‐protected 5‐carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high‐throughput manner. 相似文献
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Dr. Ivana Drienovská Remkes A. Scheele Cora Gutiérrez de Souza Prof. Gerard Roelfes 《Chembiochem : a European journal of chemical biology》2020,21(21):3077-3081
We have examined the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodology. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII-bound LmrR_HQAla enzyme was shown through its ability to catalyse asymmetric Friedel-Craft alkylation and water addition, whereas the ZnII-coupled enzyme was shown to mimic natural Zn hydrolase activity. 相似文献
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Prof. Eric Block Prof. Squire J. Booker Dr. Sonia Flores‐Penalba Prof. Graham N. George Dr. Sivaji Gundala Dr. Bradley J. Landgraf Jun Liu Dr. Stephene N. Lodge Dr. M. Jake Pushie Prof. Sharon Rozovsky Dr. Abith Vattekkatte Rama Yaghi Dr. Huawei Zeng 《Chembiochem : a European journal of chemical biology》2016,17(18):1738-1751
Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N‐(tert‐butoxycarbonyl)‐l ‐aspartic acid tert‐butyl ester. TFSeM shows enhanced methioninase‐induced cytotoxicity, relative to selenomethionine (SeM), toward HCT‐116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase. 相似文献
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Antonio Caligiuri Paola D'Arrigo Elena Rosini Davide Tessaro Gianluca Molla Stefano Servi Loredano Pollegioni 《Advanced Synthesis \u0026amp; Catalysis》2006,348(15):2183-2190
Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure. 相似文献
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Anissa Haim Dr. Saskia Neubacher Prof. Dr. Tom N. Grossmann 《Chembiochem : a European journal of chemical biology》2021,22(17):2672-2679
Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications. 相似文献
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Eger S Castrec B Hübscher U Scheffner M Rubini M Marx A 《Chembiochem : a European journal of chemical biology》2011,12(18):2807-2812
Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high‐fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono‐ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site‐specifically mono‐ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site‐specifically by the CuI‐catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA–Ub than to PCNA. 相似文献
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Modified Peptide Inhibitors of the Keap1–Nrf2 Protein–Protein Interaction Incorporating Unnatural Amino Acids
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Dr. Nikolaos D. Georgakopoulos Dr. Sandeep K. Talapatra Dr. Jemma Gatliff Prof. Frank Kozielski Dr. Geoff Wells 《Chembiochem : a European journal of chemical biology》2018,19(17):1810-1816
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Dr. Clemens Mayer 《Chembiochem : a European journal of chemical biology》2019,20(11):1357-1364
Expanding the genetic code of organisms by incorporating noncanonical amino acids (ncAAs) into target proteins through the suppression of stop codons in vivo has profoundly impacted how we perform protein modification or detect proteins and their interaction partners in their native environment. Yet, with genetic code expansion strategies maturing over the past 15 years, new applications that make use—or indeed repurpose—these techniques are beginning to emerge. This Concept article highlights three of these developments: 1) The incorporation of ncAAs for the biosynthesis and selection of bioactive macrocyclic peptides with novel ring architectures, 2) synthetic biocontainment strategies based on the addiction of microorganisms to ncAAs, and 3) enzyme design strategies, in which ncAAs with unique functionalities enable the catalysis of new-to-nature reactions. Key advances in all three areas are presented and potential future applications discussed. 相似文献
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