Faculty Profiles - MURAKAMI Hiroshi

Papers - MURAKAMI Hiroshi

Division display 41 - 60 of about 91 ／ All the affair displays >>

Ribosomal Synthesis of Cyclic Peptides with a Fluorogenic Oxidative Coupling Reaction

Yusuke Yamagishi, Hiroshi Ashigai, Yuki Goto, Hiroshi Murakami, Hiroaki Suga

ChemBioChem Vol. 10 ( 9 ) page： 1469 - 1472 2009.6

Ribosomal Synthesis of Peptides with C-Terminal Lactams, Thiolactones, and Alkylamides

Eiji Nakajima, Yuki Goto, Yusuke Sako, Hiroshi Murakami, Hiroaki Suga

ChemBioChem Vol. 10 ( 7 ) page： 1186 - 1192 2009.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

The C terminus of a peptide expressed by the translation apparatus generally ends in a carboxylate group. On the other hand, the C termini of some naturally occuring peptides have amide moieties instead of carboxylates, which are believed to give better biostability. Here, we describe a new strategy for the ribosomal synthesis of peptides featuring C-terminal lactam, thiolactone, and alkylamide units. The method was based on the concept of genetic code reprogramming involving the flexizymes (flexible tRNA acylation ribozymes) and the PURE (peptide synthesis using recombinant elements) system, in which vacant codons are reassingned to nonproteinogenic amino acids, this enabled us to convert the C termini of peptides into the above functionalities. We have also applied this method to the synthesis of a macrocyclic peptide closed by an amide bond formed between a lysine side chain and the peptide C terminus. This method thus offers us new opportunities to express various peptides using the translation apparatus, and potentially to accelerate the discovery of peptide drugs designed for various therapeutic targets.

DOI： 10.1002/cbic.200900058

Web of Science

PubMed

Bases in the anticodon loop of tRNAAlaGGC prevent misreading

Hiroshi Murakami, Atsushi Ohta, Hiroaki Suga

Nature Structural & Molecular Biology Vol. 16 ( 4 ) page： 353 - 358 2009.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

The bases at positions 32 and 38 in the tRNA anticodon loop are known to have a specific conservation depending upon the anticodon triplets. Here we report that evolutionarily conserved pairs of bases at positions 32 and 38 in tRNA(GGC)(Ala) prevent misreading of a near-cognate valine codon, GUC. The tRNA(GGC)(Ala) molecules with the conserved A32-U38 and C32-G38 pairs do not read GUC, whereas those with three representative nonconserved pairs, U32-U38, U32-A38 and C32-A38, direct the misincorporation of alanine at this valine codon into the peptide chain. Overexpression of the nonconserved tRNA(GGC)(Ala) in Escherichia coli is toxic and prevents cell growth. These results suggested that the bases at positions 32 and 38 in tRNA(GGC)(Ala) evolved to preserve the fidelity of the cognate codon reading.

DOI： 10.1038/nsmb.1580

Web of Science

PubMed

Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides

Yuki Goto, Kazuhiro Iwasaki, Kohei Torikai, Hiroshi Murakami, Hiroaki Suga

Chemical Communications ( 23 ) page： 3419 - 3419 2009

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry ({RSC})

We report here the ribosomal synthesis of methyllanthionine-containing cyclic peptides involving a site-specific incorporation of vinylglycine under the reprogrammed genetic code, followed by the isomerization of the vinylglycine to dehydrobutyrine, and the subsequent intramolecular Michael addition of a cysteine residue placed at a downstream position of the vinylglycine.

DOI： 10.1039/b904314d

Web of Science

PubMed

Ribosomal Synthesis of Polypeptoids and Peptoid−Peptide Hybrids

Takashi Kawakami, Hiroshi Murakami, Hiroaki Suga

Journal of the American Chemical Society Vol. 130 ( 50 ) page： 16861 - 16863 2008.12

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Here we report a new methodology for mRNA-programmed synthesis of peptoids and peptoid-peptide hybrids by means of translation machinery under the reprogrammed genetic code. We initially screened N-substituted glycines (rGly) for their single incorporation into a nascent peptide chain and found translation machinery accepts a variety of rGly for elongation. Moreover, we have shown consecutive elongations of rGly and mRNA-directed synthesis of cyclic peptoid-peptide hybrids. This methodology offers a powerful tool for mRNA-programmed library synthesis of peptoids and peptoid-peptide hybrids with linear and cyclic scaffolds, potentially leading to the discovery of drug candidates with proteolytic stability and membrane permeability.

DOI： 10.1021/ja806998v

Web of Science

PubMed

Polymerization of α-Hydroxy Acids by Ribosomes

Atsushi Ohta, Hiroshi Murakami, Hiroaki Suga

ChemBioChem Vol. 9 ( 17 ) page： 2773 - 2778 2008.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

Over 30 years ago, Fahnestock and Rich reported intriguing data showing the capability of the ribosome to polymerize phenyllactic acid. Although the polymerization was initiated and terminated randomly on polyuridic acids, the given data convincingly suggested that the generated polymer was composed of an approximately 7:3 mixture of phenyllactic acid and phenylalanine. Despite the fact that Fahnestock's conclusion was very likely correct, there have been no reports to follow up the ribosome-catalyzed polymerization of alpha-hydroxy acids until very recently. At the end of 2007, we reported messenger RNA (mRNA)-directed polyester synthesis by using the new emerging method of generic-code reprogramming in which alpha-hydroxy acids with various kinds of side-chains. are assigned to arbitrarily chosen codons. In this work, we have achieved the ribosomal synthesis of polyesters with the sequence composition and length in a fully controlled manner according to the sequence of mRNA. This Concept article describes the background of the method development and its application to the synthesis of polyesters.

DOI： 10.1002/cbic.200800439

Web of Science

PubMed

Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme

Hong Xiao, Hiroshi Murakami, Hiroaki Suga, Adrian R. Ferré-D’Amaré

Nature Vol. 454 ( 7202 ) page： 358 - 361 2008.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro(1-5). Flexizyme is a 45- nucleotide ribozyme capable of charging tRNA in trans with various activated L- phenylalanine derivatives. In addition to a more than 10(5) rate enhancement and more than 10(4)-fold discrimination against some non- cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH(5-7). Here we report the 2.8-angstrom resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base- pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active- site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl- tRNA synthetases(8,9) to increase specificity.

DOI： 10.1038/nature07033

Web of Science

PubMed

Initiating translation with <i>D</i>-amino acids Open Access

Yuki Goto, Hiroshi Murakami, Hiroaki Suga

RNA Vol. 14 ( 7 ) page： 1390 - 1398 2008.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Cold Spring Harbor Laboratory

<jats:p>Here we report experimental evidence that the translation initiation apparatus accepts <jats:italic>D</jats:italic>-amino acids (<jats:italic><jats:sup>D</jats:sup></jats:italic>aa), as opposed to only <jats:italic>L</jats:italic>-methionine, as initiators. Nineteen <jats:italic><jats:sup>D</jats:sup></jats:italic>aa, as the stereoisomers to their natural <jats:italic>L</jats:italic>-amino acids, were charged onto initiator tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub> using flexizyme technology and tested for initiation in a reconstituted <jats:italic>Escherichia coli</jats:italic> translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to <jats:italic><jats:sup>D</jats:sup></jats:italic>aa. Remarkably, all <jats:italic><jats:sup>D</jats:sup></jats:italic>aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with <jats:italic><jats:sup>D</jats:sup></jats:italic>aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding <jats:italic><jats:sup>D</jats:sup></jats:italic>aa-tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub>. Although the inefficient formylation of <jats:italic><jats:sup>D</jats:sup></jats:italic>aa-tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub> resulted in modest expression of the corresponding peptide, preacetylation of <jats:italic><jats:sup>D</jats:sup></jats:italic>aa-tRNA<jats:sup>fMet</jats:sup><jats:sub>CAU</jats:sub> dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with <jats:italic><jats:sup>D</jats:sup></jats:italic>aa. Our findings provide not only the experimental evidence that translation initiation tolerates <jats:italic><jats:sup>D</jats:sup></jats:italic>aa, but also a new means for the mRNA-directed synthesis of peptides capped with <jats:italic><jats:sup>D</jats:sup></jats:italic>aa or acyl-<jats:italic><jats:sup>D</jats:sup></jats:italic>aa at the N terminus.</jats:p>

DOI： 10.1261/rna.1020708

Web of Science

PubMed

Ribosomal Synthesis of Bicyclic Peptides via Two Orthogonal Inter-Side-Chain Reactions

Yusuke Sako, Jumpei Morimoto, Hiroshi Murakami, Hiroaki Suga

Journal of the American Chemical Society Vol. 130 ( 23 ) page： 7232 - 7234 2008.6

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Here we report a new methodology for the synthesis of bicyclic peptides by using a reconstituted cell-free translation system under the reprogrammed genetic code. Cysteine (Cys) and three different nonproteinogenic amino acids, Cab, Aha, and PgI, were simultaneously incorporated into a peptide chain. The first cyclization occurred between the chloroacetyl group of Cab and the sulfhydryl group in Cys in situ of translation, and the second cyclization on the side chains of Aha-Pgl via Cu(I)-catalyzed azide-alkyne cycloaddition was performed. This offers us a powerful means of mRNA-programmed synthesis of various peptides with uniform bicyclic scaffolds.

DOI： 10.1021/ja800953c

Web of Science

PubMed

Ribosomal Synthesis of Peptidase-Resistant Peptides Closed by a Nonreducible Inter-Side-Chain Bond

Yusuke Sako, Yuki Goto, Hiroshi Murakami, Hiroaki Suga

ACS Chemical Biology Vol. 3 ( 4 ) page： 241 - 249 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Here we report a new enabling technology for the synthesis of peptidase-resistant cyclic peptides by means of genetic code reprogramming involving the flexizyme (a tRNA acylation ribozyme) and PURE (a reconstituted cell-free translation) systems. in this work, we have developed a new nonproteinogenic amino acid bearing a chloroacetyl group in the side chain, which forms a physiologically stable thioether bond by intramolecular reaction with the sulfhydryl group of a Cys residue in the peptide chain upon translation. Significantly, this chemistry takes place spontaneously in situ of the translation solution, giving the corresponding cyclic peptides independent of ring sizes. We have used this method to convert human urotensin 11, known as a potent vasoconstrictor, to its analogue containing a thioether bond, showing that this new analogue retains biological activity. Moreover, this peptide exhibits remarkable resistance against peptidases under reducing conditions. Thus, this technology offers a new means to accelerate the discovery of therapeutic peptidic drugs.

DOI： 10.1021/cb800010p

Web of Science

PubMed

Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides Reviewed

Yuki Goto, Atsushi Ohta, Yusuke Sako, Yusuke Yamagishi, Hiroshi Murakami, Hiroaki Suga

ACS CHEMICAL BIOLOGY Vol. 3 ( 2 ) page： 120 - 129 2008.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report here a new technology to reprogram the initiation event, where various amino acids and those bearing N-alpha-acyl groups can be used as an initiator for peptide synthesis. The technology is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technology to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiologically stable bond, and it is also extended to the custom synthesis of its analogues with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodology, we also prepared a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technology offers a new means to prepare a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.

DOI： 10.1021/c6700233t

Web of Science

PubMed

Messenger RNA-Programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides Reviewed

Takashi Kawakami, Hiroshi Murakami, Hiroaki Suga

CHEMISTRY & BIOLOGY Vol. 15 ( 1 ) page： 32 - 42 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：CELL PRESS

Natural peptide products often contain N-methylated backbones, and such a modification plays a crucial role in making natural peptides peptidase resistant and membrane permeable. Here, we demonstrate the ribosomal synthesis of N-methyl-peptides by means of genetic code reprogramming. Two key technologies, a ribozyme-based de novo tRNA acylation (flexizyme) system and an E coli reconstituted cell-free translation (PURE) system, were used in order to reassign arbitrarily chosen codons to N-alpha-methylated amino acids ((Me)aa). Using this combination, we determined the general structural requirement of "accessible" Meaa and demonstrated their multiple incorporations into the nascent peptide chain according to the assignments made on mRNA, giving linear and cyclic N-methyl-peptides in high purities. This platform technology offers a convenient tool for the construction of N-methyl-peptide libraries, potentially leading to the discovery of therapeutic peptides.

DOI： 10.1016/j.chembiol.2007.12.008

Web of Science

PubMed

Synthesis of Polyester by Means of Genetic Code Reprogramming

Atsushi Ohta, Hiroshi Murakami, Eri Higashimura, Hiroaki Suga

Chemistry & Biology Vol. 14 ( 12 ) page： 1315 - 1322 2007.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

Here we report the ribosomal polymerization of alpha-hydroxy acids by means of genetic code reprogramming. The flexizyme system, a ribozyme-based tRNA acylation tool, was used to reassign individual codons to seven types of a-hydroxy acids, and then polyesters were synthesized under controls of the reprogrammed genetic code using a reconstituted cell-free translation system. The sequence and length of the polyester segments were specified by the mRNA template, indicating that high-fidelity ribosome expression of polyesters was possible. This work opens a door for the mRNA-directed synthesis of backbone-altered biopolymers.

DOI： 10.1016/j.chembiol.2007.10.015

Web of Science

PubMed

The flexizyme system: a highly flexible tRNA aminoacylation tool for the translation apparatus

Masaki Ohuchi, Hiroshi Murakami, Hiroaki Suga

Current Opinion in Chemical Biology Vol. 11 ( 5 ) page： 537 - 542 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

Flexizymes are de novo ribozymes capable of charging a wide variety of non-natural amino acids on tRNAs. The flexizyme system enables reprogramming of the genetic code by reassigning the codons that are generally assigned to natural amino acids to non-natural residues, and thus mRNA-directed synthesis of non-natural polypeptides can be achieved. In this review, we comprehensively summarize the history of the flexizyme system and its subsequent development into a practical tool. Furthermore, applications to the synthesis of novel biopolymers via genetic code reprogramming and perspectives for future applications are described.

DOI： 10.1016/j.cbpa.2007.08.011

Web of Science

PubMed

[Reprogramming the genetic code using flexizyme].

Sako Y, Murakami H, Suga H

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Vol. 52 ( 13 Suppl ) page： 1643 - 8 2007.10

Ribosomal Polyester Synthesis Using Reprogrammed Genetic Code Reviewed

Atsushi Ohta, Hiroshi Murakami, Hiroaki Suga

Kobunshi Vol. 56 ( 4 ) page： 196 - 199 2007

Language：English Publishing type：Research paper (scientific journal)

The translation apparatus is sophisticated machinery that polymerizes amino acids to polypeptide according to the genetic triplets encoded on messenger RNA (mRNA) sequence. Because of this property, ribosome generally acts as a superb template-directed polymer synthesizer and therefore translated polypeptide shows narrow distribution of molecular weights with complete controlled sequence. Unfortunately, this superior system is limited to polypeptide synthesis because of the lack of genetic code which converts genetic triplets to any monomers except for 20 natural amino acids. To expand the application of ribosome, we utilized the concept of genetic code reprogramming to assign several codons as hydroxy acids and performed mRNA-templated polyester synthesis. We have demonstrated the sequence and length of polyesters can be controlled by the mRNA sequence as designed. This is a novel technology for the synthesis of polyester and polyester-polypeptide copolymer for the investigation of new materials. © 2007, The Society of Polymer Science, Japan. All rights reserved.

DOI： 10.1295/kobunshi.56.196

Scopus

In situ generation of aminoacyl-tRNAs assisted by ribozymes in translation apparatus. Reviewed

Ohuchi M, Murakami H, Suga H

Nucleic acids symposium series (2004) ( 51 ) page： 115 - 116 2007

Exploration of incorporation of Nalpha-methylated amino acids into peptides by sense-suppression method. Reviewed

Kawakami T, Murakami H, Suga H

Nucleic acids symposium series (2004) ( 51 ) page： 361 - 362 2007

[Expansion and reprogramming of genetic code by Flexizyme].

Murakami H, Suga H

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme Vol. 51 ( 16 Suppl ) page： 2496 - 501 2006.12

フレキシザイムを用いた遺伝暗号の拡張とリプログラミング (RNAと生命) -- (RNAテクノロジーと創薬)

村上裕, 菅裕明

蛋白質核酸酵素 Vol. 51 ( 16 ) page： 2496-2501 2006.12

　 PREV - NEXT 　

<MURAKAMI Hiroshi>

Papers - MURAKAMI Hiroshi