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1.
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
2.
Two‐Tier Screening Platform for Directed Evolution of Aminoacyl–tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency
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Andrew E. Owens Katherine T. Grasso Christine A. Ziegler Prof. Rudi Fasan 《Chembiochem : a European journal of chemical biology》2017,18(12):1109-1116
Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non‐proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl–tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate‐based colorimetric assay, thereby combining high‐throughput capabilities with reliable and quantitative measurement of aaRS‐dependent ncAA incorporation efficiency. This two‐tier functional screening system was successfully applied to obtain a pyrrolysyl–tRNA synthetase (PylRS) variant (CrtK‐RS(4.1)) with significantly improved efficiency (+250–370 %) for mediating the incorporation of N?‐crotonyl‐lysine and other lysine analogues of relevance for the study of protein post‐translational modifications into a target protein. Interestingly, the beneficial mutations accumulated by CrtK‐RS(4.1) were found to localize within the noncatalytic N‐terminal domain of the enzyme and could be transferred to another PylRS variant, improving the ability of the variant to incorporate its corresponding ncAA substrate. This work introduces an efficient platform for the improvement of aaRSs that could be readily extended to other members of this enzyme family and/or other target ncAAs. 相似文献
3.
Probing the Outstanding Local Hydrophobicity Increases in Peptide Sequences Induced by Incorporation of Trifluoromethylated Amino Acids
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Dr. Charlène Gadais Dr. Emmanuelle Devillers Dr. Vincent Gasparik Dr. Evelyne Chelain Dr. Julien Pytkowicz Prof. Dr. Thierry Brigaud 《Chembiochem : a European journal of chemical biology》2018,19(10):1026-1030
In order to achieve accurate determination of the local hydrophobicity increases in peptide sequences produced by incorporation of trifluoromethylated amino acids (TfmAAs), the chromatographic hydrophobicity indexes (?0) of three series of tripeptides containing three unnatural trifluoromethylated amino acids have been measured and compared with those of their non‐fluorinated analogues. The hydrophobic contribution of each fluorinated amino acid was quantified by varying the position and the protection of (R)‐ and (S)‐α‐trifluoromethylalanine (TfmAla), (R)‐trifluoromethylcysteine (TfmCys), and (S)‐trifluoromethionine (TFM) in a short peptide sequence. As a general trend, strong increases in hydrophobicity were precisely measured, even exceeding the high hydrophobic contribution of the natural amino acid isoleucine. This study validates the incorporation of trifluoromethylated amino acids into peptide sequences as a rational strategy for the fine‐tuning of hydrophobic peptide–protein interactions. 相似文献
4.
Biosynthetic Gene Cluster for Surugamide A Encompasses an Unrelated Decapeptide,Surugamide F
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Akihiro Ninomiya Prof. Yohei Katsuyama Prof. Takefumi Kuranaga Masayuki Miyazaki Dr. Yuichi Nogi Prof. Shigeru Okada Prof. Toshiyuki Wakimoto Prof. Yasuo Ohnishi Prof. Shigeki Matsunaga Prof. Kentaro Takada 《Chembiochem : a European journal of chemical biology》2016,17(18):1709-1712
Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A–E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non‐ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A–E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3‐amino‐2‐methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid‐phase peptide synthesis. 相似文献
5.
Zhi Feng Dr. Yasushi Ogasawara Satoshi Nomura Prof. Dr. Tohru Dairi 《Chembiochem : a European journal of chemical biology》2018,19(19):2045-2048
MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271. 相似文献
6.
Hydrophilic trans‐Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling
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Eszter Kozma Dr. Ivana Nikić Dr. Balázs R. Varga Iker Valle Aramburu Jun Hee Kang Dr. Oliver T. Fackler Dr. Edward A. Lemke Dr. Péter Kele 《Chembiochem : a European journal of chemical biology》2016,17(16):1518-1524
Introduction of bioorthogonal functionalities (e.g., trans‐cyclooctene‐TCO) into a protein of interest by site‐specific genetic encoding of non‐canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine‐functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours‐long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO‐ncAAs. One derivative, DOTCO‐lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy. 相似文献
7.
Biosynthesis of the Peptide Antibiotic Feglymycin by a Linear Nonribosomal Peptide Synthetase Mechanism
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Melanie Gonsior Dr. Agnes Mühlenweg Marcel Tietzmann Dr. Saskia Rausch Annette Poch Prof. Dr. Roderich D. Süssmuth 《Chembiochem : a European journal of chemical biology》2015,16(18):2610-2614
Feglymycin, a peptide antibiotic produced by Streptomyces sp. DSM 11171, consists mostly of nonproteinogenic phenylglycine‐type amino acids. It possesses antibacterial activity against methicillin‐resistant Staphylococcus aureus strains and antiviral activity against HIV. Inhibition of the early steps of bacterial peptidoglycan synthesis indicated a mode of action different from those of other peptide antibiotics. Here we describe the identification and assignment of the feglymycin (feg) biosynthesis gene cluster, which codes for a 13‐module nonribosomal peptide synthetase (NRPS) system. Inactivation of an NRPS gene and supplementation of a hydroxymandelate oxidase mutant with the amino acid l ‐Hpg proved the identity of the feg cluster. Feeding of Hpg‐related unnatural amino acids was not successful. This characterization of the feg cluster is an important step to understanding the biosynthesis of this potent antibacterial peptide. 相似文献
8.
Emma J. Rackham Sabine Grüschow Dr. Amany E. Ragab Shilo Dickens Dr. Rebecca J. M. Goss Dr. 《Chembiochem : a European journal of chemical biology》2010,11(12):1700-1709
The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3′‐deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact. 相似文献
9.
John J. Barkei Brian M. Kevany Dr. Elizabeth A. Felnagle Michael G. Thomas Prof. 《Chembiochem : a European journal of chemical biology》2009,10(2):366-376
Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted‐gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with β‐lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the β‐amino group of L ‐2,3‐diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and capreomycin. 相似文献
10.
Benjamin Philmus Guntram Christiansen Dr. Wesley Y. Yoshida Thomas K. Hemscheidt Prof. 《Chembiochem : a European journal of chemical biology》2008,9(18):3066-3073
Cyanobacteria are prolific producers of bioactive natural products that mostly belong to the nonribosomal peptide and polyketide classes. We show here how a linear precursor peptide of microviridin K, a new member of the microviridin class of peptidase inhibitors, is processed to become the mature tricyclic peptidase inhibitor. The microviridin (mvd) biosynthetic gene cluster of P. agardhii comprises six genes encoding microviridin K, an apparently unexpressed second microviridin, two RimK homologues, an acetyltransferase, and an ABC transporter. We have over‐expressed three enzymes of this pathway and have demonstrated their biochemical function in vitro through chemical degradation and mass spectrometry. We show that a prepeptide undergoes post‐translational modification through cross‐linking by ester and amide bond formation by the RimK homologues MvdD and MvdC, respectively. In silico analysis of the mvd gene cluster suggests the potential for widespread occurrence of microviridin‐like compounds in a broad range of bacteria. 相似文献
11.
Shiyu Chen Dr. David Gfeller Sergey A. Buth Prof. Olivier Michielin Prof. Petr G. Leiman Prof. Christian Heinis 《Chembiochem : a European journal of chemical biology》2013,14(11):1316-1322
Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied. 相似文献
12.
Arginine Arrangement of Bacteriophage λ N‐Peptide Plays a Role as a Core Motif in GNRA Tetraloop RNA Binding
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Prof. Dr. Hiroyuki Furusawa Dr. Shinobu Fukusho Prof. Dr. Yoshio Okahata 《Chembiochem : a European journal of chemical biology》2014,15(6):865-871
A simple α‐helical N‐model‐peptide was designed to investigate the role of the arginine‐rich motif of bacteriophage λ N‐peptide in selective binding with boxB RNA. The five‐arginine arrangement of native N‐peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random‐nucleotide region) was carried out with N‐model‐peptide immobilized on a 27 MHz quartz‐crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (Ka=107–108 M ?1). Many selected RNAs contained GNR(N)A‐type loops (similar to the boxB RNA motif recognized by the native N‐peptide). Fragments and minimal RNAs containing the GNRA‐type loop also bound to N‐model‐peptide (Ka=106–107 M ?1). The RNA recognition specificity of the peptide was studied by changing the “closing” U–A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N‐model‐peptide). It was concluded that the five‐arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N‐peptide adds the recognition ability for a loop‐RNA sequence. 相似文献
13.
Polyimides (PIs) as high‐performance organic matrices are used in the preparation of PI composites because of their excellent mechanical, thermal and dielectric properties. The sol–gel method is a promising technique for preparing these PI composites due to the mild reaction conditions and the process being controllable. Although sulfonated polyimide (SPI) proton‐exchange membranes have attracted much attention recently, studies on preparing SPI‐based hybrid proton‐exchange membranes for fuel cells have been rare. A series of SPI? SiO2 hybrid proton‐exchange membranes were prepared from amino‐terminated SPI pre‐polymers, 3‐glycidoxypropyltrimethoxysilane (KH‐560) and tetraethylorthosilicate through a co‐hydrolysis and condensation process using an in situ sol–gel method. The reactive silane KH‐560 was used to react with amino‐terminated SPI to form silane‐capped SPI in order to improve the compatibility between the polymer matrix and the inorganic SiO2 phase. The microstructure and mechanical, thermal and proton conduction properties were studied in detail. The hybrid membranes were highly uniform without phase separation up to 30 wt% SiO2. The storage modulus and tensile strength of the hybrid membranes increased with increasing SiO2 content. The introduction of SiO2 improved the methanol resistance while retaining good proton conductivity. The hybrid membrane with 30 wt% SiO2 exhibited a proton conductivity of 10.57 mS cm?1 at 80 °C and methanol permeability of 2.3 × 10?6 cm2 s?1 possibly because the crosslinking structure and SiO2 phases formed in the hybrids could retain water and were helpful to proton transport. Copyright © 2010 Society of Chemical Industry 相似文献
14.
Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins
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Dr. Sophia Zobel Simon Boecker Dr. Daniel Kulke Dr. Dirk Heimbach Prof. Dr. Vera Meyer Prof. Dr. Roderich D. Süssmuth 《Chembiochem : a European journal of chemical biology》2016,17(4):283-287
Non‐ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa‐ and cyclo‐octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy‐acid‐activating module of PF1022 synthetase with the amino‐acid‐activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity. 相似文献
15.
Olubodun Michael Lateef Michael Olawale Akintubosun Olamide Tosin Olaoba Sunday Ocholi Samson Malgorzata Adamczyk 《International journal of molecular sciences》2022,23(2)
The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications. 相似文献
16.
Jürgen Rohr Prof. Dr. 《Chembiochem : a European journal of chemical biology》2010,11(4):523-532
The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis. 相似文献
17.
Dr. Fumihiro Ishikawa Prof. Genzoh Tanabe 《Chembiochem : a European journal of chemical biology》2019,20(16):2032-2040
Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme–substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment. 相似文献
18.
Soy protein has been considered as an alternative to partly replace petroleum‐based polymers for adhesive applications. The weakness of protein‐based adhesive is poor water resistance, which limits its outdoor applications. The objective of this research was to improve the water resistance of soy protein adhesive by introducing crosslinkage between amino groups of amino acid residue. Laboratory prepared soy protein isolate (SPI) was used in this study. Glutaraldehyde at concentrations of 4, 20, 40, and 80 μM was used as the crosslinking reagent for SPI modification. Adhesive properties of soy protein modified by glutaraldehyde, as well as thermal and morphological properties, were investigated. Crosslinking‐induced protein conformation and structure changes through decrease of amino groups and adding of hydrophobic groups, subsequently affect adhesive performance of SPI. At optimum glutaraldehyde concentration (20 μM), dry, wet, and soak strengths of modified SPI increased to 31.5, 115, and 29.7%, respectively, compared with unmodified SPI. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 130–136, 2007 相似文献
19.
Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein's gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a "pulse" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology. 相似文献
20.
Advanced Mutasynthesis Studies on the Natural α‐Pyrone Antibiotic Myxopyronin from Myxococcus fulvus
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J. Henning Sahner Hilda Sucipto Dr. Silke C. Wenzel Dr. Matthias Groh Prof. Dr. Rolf W. Hartmann Prof. Dr. Rolf Müller 《Chembiochem : a European journal of chemical biology》2015,16(6):946-953
Myxopyronin is a natural α‐pyrone antibiotic from the soil bacterium Myxococcus fulvus Mx f50. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by binding to a part of the enzyme not targeted by the clinically used rifamycins. This mode of action makes myxopyronins promising molecules for the development of novel broad‐spectrum antibacterials. We describe the derivatization of myxopyronins by an advanced mutasynthesis approach as a first step towards this goal. Site‐directed mutagenesis of the biosynthetic machinery was used to block myxopyronin biosynthesis at different stages. The resulting mutants were fed with diverse precursors that mimic the biosynthetic intermediates to restore production. Mutasynthon incorporation and production of novel myxopyronin derivatives were analyzed by HPLC‐MS/MS. This work sets the stage for accessing numerous myxopyronin derivatives, thus significantly expanding the chemical space of f α‐pyrone antibiotics. 相似文献