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Dive into the research topics where Koichiro Kodama is active.

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Featured researches published by Koichiro Kodama.


Nature Biotechnology | 2002

An unnatural base pair for incorporating amino acid analogs into proteins

Ichiro Hirao; Takashi Ohtsuki; Tsuyoshi Fujiwara; Tsuneo Mitsui; Tomoko Yokogawa; Taeko Okuni; Hiroshi Nakayama; Koji Takio; Takashi Yabuki; Takanori Kigawa; Koichiro Kodama; Takashi Yokogawa; Kazuya Nishikawa; Shigeyuki Yokoyama

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography–mass spectrometry (LC-MS) analysis of the products. This coupled transcription–translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.


Proceedings of the National Academy of Sciences of the United States of America | 2002

An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system

Daisuke Kiga; Kensaku Sakamoto; Koichiro Kodama; Takanori Kigawa; Takayoshi Matsuda; Takashi Yabuki; Mikako Shirouzu; Yoko Harada; Hiroshi Nakayama; Koji Takio; Yoshinori Hasegawa; Yaeta Endo; Ichiro Hirao; Shigeyuki Yokoyama

Tyrosyl–tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-l-tyrosine rather than l-tyrosine for the site-specific incorporation of 3-iodo-l-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-l-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the l-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-l-tyrosine 10-fold more efficiently than l-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNATyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-l-tyrosine. After the use of the 3-iodotyrosyl–tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of l-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-l-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.


ChemBioChem | 2006

Regioselective Carbon–Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic Chemistry

Koichiro Kodama; Seketsu Fukuzawa; Hiroshi Nakayama; Takanori Kigawa; Kensaku Sakamoto; Takashi Yabuki; Natsuko Matsuda; Mikako Shirouzu; Koji Takio; Kazuo Tachibana; Shigeyuki Yokoyama

Palladium‐catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki–Heck reaction for site‐specific carbon–carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6‐fused Ras protein containing 4‐iodo‐L‐phenylalanine at position 32 (iF32‐Ras‐His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32‐Ras‐His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography–mass spectrometry (LC‐MS). The regioselectivity of the Mizoroki–Heck reaction was furthermore confirmed by LC‐MS/MS analysis. However, in addition to the biotinylated product (bF32‐Ras‐His), a dehalogenated product (F32‐Ras‐His) was detected by LC‐MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki–Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras‐binding domain as its downstream target, Raf‐1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.


ChemBioChem | 2007

Site‐Specific Functionalization of Proteins by Organopalladium Reactions

Koichiro Kodama; Seketsu Fukuzawa; Hiroshi Nakayama; Kensaku Sakamoto; Takanori Kigawa; Takashi Yabuki; Natsuko Matsuda; Mikako Shirouzu; Koji Takio; Shigeyuki Yokoyama; Kazuo Tachibana

A new carbon–carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki–Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild‐type Ras protein. Finally, the iF–Ras proteins containing a 4‐iodo‐L‐phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site‐specific biotinylations of the Ras protein were confirmed by Western blot and LC‐MS/MS.


Bioorganic & Medicinal Chemistry Letters | 2008

N-terminal labeling of proteins by the Pictet–Spengler reaction

Tsubasa Sasaki; Koichiro Kodama; Hiroaki Suzuki; Seketsu Fukuzawa; Kazuo Tachibana

The Pictet-Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an alpha-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet-Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet-Spengler reaction.


ChemBioChem | 2006

A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe.

Koichiro Kodama; Seketsu Fukuzawa; Kensaku Sakamoto; Hiroshi Nakayama; Takanori Kigawa; Takashi Yabuki; Natsuko Matsuda; Mikako Shirouzu; Koji Takio; Kazuo Tachibana; Shigeyuki Yokoyama

An Escherichia coli suppressor tRNAPhe (tRNAPheCUA) was misacylated with 4‐iodo‐L‐phenylalanine by using the A294G phenylalanyl–tRNA synthetase mutant (G294‐PheRS) from E. coli at a high magnesium‐ion concentration. The preacylated tRNA was added to an E. coli cell‐free system and a Ras protein that contained the 4‐iodo‐L‐phenylalanine residue at a specific target position was synthesized. Site‐specific incorporation of 4‐iodo‐L‐phenylalanine was confirmed by using LC–MS/MS. Free tRNAPheCUA was not aminoacylated by aminoacyl–tRNA synthetases (aaRSs) present in the E. coli cell‐free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site‐specific functionalization of proteins.


ChemBioChem | 2015

Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation

Haruhiko Ehara; Marie Makino; Koichiro Kodama; Keiichi Konoki; Takuhiro Ito; Shun-ichi Sekine; Seketsu Fukuzawa; Shigeyuki Yokoyama; Kazuo Tachibana

Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α‐helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.


Journal of Biochemistry | 2010

Site-specific incorporation of 4-Iodo-l-phenylalanine through opal suppression

Koichiro Kodama; Hiroshi Nakayama; Kensaku Sakamoto; Seketsu Fukuzawa; Takanori Kigawa; Takashi Yabuki; Makoto Kitabatake; Koji Takio; Shigeyuki Yokoyama

A variety of unique codons have been employed to expand the genetic code. The use of the opal (UGA) codon is promising, but insufficient information is available about the UGA suppression approach, which facilitates the incorporation of non-natural amino acids through suppression of the UGA codon. In this study, the UGA codon was used to incorporate 4-iodo-l-phenylalanine into position 32 of the Ras protein in an Escherichia coli cell-free translation system. The undesired incorporation of tryptophan in response to the UGA codon was completely repressed by the addition of indolmycin. The minor amount (3%) of contaminating 4-bromo-l-phenylalanine in the building block 4-iodo-l-phenylalanine led to the significant incorporation of 4-bromo-l-phenylalanine (21%), and this problem was solved by using a purified 4-iodo-l-phenylalanine sample. Optimization of the incubation time was also important, since the undesired incorporation of free phenylalanine increased during the cell-free translation reaction. The 4-iodo-l-phenylalanine residue can be used for the chemoselective modification of proteins. This method will contribute to advancements in protein engineering studies with non-natural amino acid substitutions.


Journal of Structural and Functional Genomics | 2001

Selenomethionine incorporation into a protein by cell-free synthesis.

Takanori Kigawa; Emi Yamaguchi-Nunokawa; Koichiro Kodama; Takayoshi Matsuda; Takashi Yabuki; Natsuko Matsuda; Ryuichiro Ishitani; Osamu Nureki; Shigeyuki Yokoyama


ChemBioChem | 2006

Cover Picture: Regioselective Carbon–Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic Chemistry (ChemBioChem 1/2006)

Koichiro Kodama; Seketsu Fukuzawa; Hiroshi Nakayama; Takanori Kigawa; Kensaku Sakamoto; Takashi Yabuki; Natsuko Matsuda; Mikako Shirouzu; Koji Takio; Kazuo Tachibana; Shigeyuki Yokoyama

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Shigeyuki Yokoyama

National Institute of Advanced Industrial Science and Technology

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Hiroshi Nakayama

Tokyo University of Agriculture and Technology

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Takanori Kigawa

Tokyo Institute of Technology

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Kensaku Sakamoto

Massachusetts Institute of Technology

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Mikako Shirouzu

National Institute of Advanced Industrial Science and Technology

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