Ibaraki University's
College of Engineering
Department of Biomolecular Functional Engineering


KIMURA Shigenobu

Other affiliation / position

  1. Frontier Research Center for Applied Atomic Sciences


  1. Reseacher, Basic Research Laboratories, Toray Industries Inc. 1984/04-1986/03
  2. Reseacher, Protein Engineering Resarch Institute (PERI) 1986/04-1992/03
  3. Reseach Scientist, Basic Research Laboratories, Toray Industries Inc. 1992/04-1996/09
  4. Associate Professor, Department of Life Science, Faculty of Science, Himeji Institute of Technology 1996/10-2002/03
  5. Associate Professor, Department of Life Science, Graduate School of Science, Himeji Institute of Technology 2002/04-2004/03
  6. Associate Professor, Graduate School of Life Science, University of Hyogo 2004/04-2005/03
  7. Professor, Department of Biomolecular Functional Engineering, Faculty of Engineering, Ibaraki University 2005/04-Present

Academic background

  1. Tohoku University 理学研究科  化学第二専攻 博士課程前期2年の過程 1984 Graduated
  2. Tohoku University Faculty of Science Department of Chemistry 1982 Graduated Japan
  3. Graduate School of Tohoku University Department of Science Chemistry Master course 1984 Completed

Academic degrees

  1. Ph.D. Tohoku University 1994/01

Current state of research and teaching activities

Structure and function of electron transfer proteins
Basic and applied research on protein engineering

Research Areas

  1. Structural Biochemistry
  2. Functional Biochemistry
  3. Environmental Technology/Environmental Material

Research keywords

  1. protein, protein engineering, electron transfer, flavoenzyme, expression system, bioremediation

Subject of research

  1. Structure and function of electron transfer protein
  2. Gene expression system for production of recombinant proteins
  3. 環境分野への応用を目指した蛋白質工学的研究 電子伝達系酵素の基質特異性改変を行い、これをバイオレメディエーションに応用する。


  1. 貧栄養下で芳香族化合物を分解できる新規光合成微生物 西澤明人、千田美紀、千田俊哉、菓子野康浩、福田雅夫、木村成伸 ケミカルエンジニヤリング 2009/10/01
  2. Molecular Mechanism of the Redox-dependent Interaction betweenNADH-dependent Ferredoxin Reductase and Rieske-type Ferredoxin Miki Senda, Shigenobu Kimura, Masao Fukuda, Tetsuo Ishida, Toshiya Senda J. Cryst. Soc. Jpn. 2008/12 Redox-dependent affinity regulation is critical to fast and efficient electron transfer(ET)between ET proteins. The molecular mechanism of the affinity regulation, however, remains elusive due to the lack of crystal structures of the ET proteins in every redox state relevant to the ET reaction. BphA4 and BphA3 are, respectively, an FAD-containing NADH-dependent ferredoxin reductase and a Rieske-type[2Fe-2S]ferredoxin of a biphenyl dioxygenase BphA derived from Acidovorax sp. strain KKS102. Our biochemical study showed that the reduction of the FAD in BphA4 increases the affinity between BphA3 and BphA4 approximately 20-fold. In order to reveal the molecular mechanism of this redox-dependent affinity regulation, we determined the crystal structure of the following molecular species: BphA4 in oxidized, hydroquinone, semiquinone, and reoxidized forms; BphA3 in oxidized and reduced forms; and the ET complex of BphA3 and BphA4. A comparative analysis of these seven crystal structures obtained revealed that a series of conformational changes of BphA4 occurs upon reduction of FAD to form a high-affinity BphA3-binding site in BphA4.
  3. Molecular Mechanism of the Redox-dependent Interaction between NADH-dependent Ferredoxin Reductase and Rieske-type [2Fe-2S] Ferredoxin Miki SENDA, Shigenobu KIMURA, Masao FUKUDA, Tetso ISHIDA, Toshiya SENDA 化学と生物 2008/10


  1. Research paper (scientific journal) Joint Complete pyridine nucleotide-specificity conversion of an NADH-dependent ferredoxin reductase. Akito Nishizawa, Ayaka Harada, Miki Senda, Yuka Tachihara, Daisuke Muramatsu, Shinya Kishigami, Shigemasa Mori, Keisuke Sugiyama, Toshiya Senda, shigenobu Kimura Biochem. J. 462/ 2, 257-265 2014/09/01 10.1042/BJ20140384 The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme to an NADPH-specific one is a useful approach to the efficient carbon neutral production of biological compounds in photosynthetic organisms. In this study, an NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu175-Thr176-Gln177 of an NADH recognition loop in the wild-type BphA4 was replaced with Cys175-Arg176-Gly177, was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.
  2. Research paper (scientific journal) Joint Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells Akito Nishizawa, Miki Nakayama, Takuya Uemura, Yoshiyuki Fukuda, and Shigenobu Kimura J. Biochem. 147/ 3, 433-443 2010/03 10.1093/jb/mvp187 Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b5 (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested (Kimura, S. et al. (2005) J. Biochem. 137, 523–533). In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD-sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD-sequence, increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation, but also for increasing the soluble form of recombinant proteins in E. coli cells.
  3. (MISC) Introduction and explanation (scientific journal) Joint Redox control of protein conformation in flavoproteins. Toshiya Senda, Miki Senda, Shigenobu Kimura, Tetsuo Ishida Antioxidants & Redox Signaling 11/ 7, 1741-1766 2009 10.1089 Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are two flavin prosthetic groups utilized as the redox centers of various proteins. The conformations and chemical properties of these flavins can be affected by their redox states as well as by photoreactions. Thus, proteins containing flavin (flavoproteins) can function not only as redox enzymes, but also as signaling molecules by using the redox- and/or light-dependent changes of the flavin. Redox and light-dependent conformational changes of flavoproteins are critical to many biological signaling systems. In this review, we summarize the molecular mechanisms of the redox-dependent conformational changes of flavoproteins and discuss their relationship to signaling functions. The redox-dependent (or light-excited) changes of flavin and neighboring residues in proteins act as molecular gswitchesh that gturn onh various conformational changes in proteins, and can be classified into five types. On the basis of the present analysis, we recommend future directions in molecular structural research on flavoenzymes and related proteins.
  4. Research paper (scientific journal) Joint Molecular mechanism of the redox-dependent interaction between NADH-dependent ferredoxin reductase and Rieske-type [2Fe-2S] ferredoxin. Miki Senda, Shinya Kishigami, Shigenobu Kimura, Masao Fukuda, Tetsuo Ishida, Toshiya Senda J.Mol.Biol. 373/ 2, 382-400 2007/10/19 10.1016/j.jmb.2007.08.002 The electron transfer system of the biphenyl dioxygenase BphA, which is derived from Acidovorax sp. (formally Pseudomonas sp.) strain KKS102, is composed of a FAD-containing NADH-ferredoxin reductase (BphA4) and a Rieske-type [2Fe-2S] ferredoxin (BphA3). Biochemical studies have suggested that the whole electron transfer process from NADH to BphA3 comprises three consecutive elementary electron-transfer reactions, in which BphA3 and BphA4 interact transiently in a redox-dependent manner. Initially, BphA4 receives two electrons from NADH. The reduced BphA4 then delivers one electron each to the [2Fe-2S] cluster of the two BphA3 molecules through redox-dependent transient interactions. The reduced BphA3 transports the electron to BphA1A2, a terminal oxygenase, to support the activation of dioxygen for biphenyl dihydroxylation. In order to elucidate the molecular mechanisms of the sequential reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3-BphA4 complex, and of free BphA3 and BphA4 in all the redox states occurring in the catalytic cycle. The crystal structures of these reaction intermediates demonstrated that each elementary electron transfer induces a series of redox-dependent conformational changes in BphA3 and BphA4, which regulate the interaction between them. In addition, the conformational changes induced by the preceding electron transfer seem to induce the next electron transfer. The interplay of electron transfer and induced conformational changes seems to be critical to the sequential electron-transfer reaction from NADH to BphA3.
  5. Research paper (scientific journal) Joint Two-citronic expression plasmids for high-level gene expression in Escherichia coli preventing translational initiation inhibition caused by the intramolecular local secondary structure of mRNA. Shigenobu Kimura, Tomoka Umemura, Takashi Iyanagi J. Biochem. 137/ 4, 523-533 2005 大腸菌での異種蛋白質遺伝子発現において、mRNAのリボソーム結合部位の分子内二次構造による翻訳阻害阻害を回避する目的で、2-シストロン性の高発現プラスミドpCP1及びpCP2を作製し、ブタ肝臓P450還元酵素遺伝子等を用いて、その有用性を確認した。

Research presentations

  1. Poster presentation Biphenyl degradation by recombinant cyanobacterium Synechocystis sp. PCC6803 in oligotrophic environment 19th International Symposium on Flavins and Flavoproteins 2017/07/02
  2. Poster presentation PetAのSec型シグナル様配列を用いたシアノバクテリアチラコイド膜画分への異種タンパクの蓄積 第17回日本蛋白質科学会年会 2017/06/21
  3. Poster presentation Accumulation of the recombinant proteins in the thylakoid membrane fraction of cyanobacteria The 12th International Student Conference at Ibaraki University (ISCIU) 2016/12/17
  4. Poster presentation シアノバクテリアチラコイド膜画分への遺伝子組換え型タンパクの蓄積 第27回日本化学会関東支部茨城地区研究交流会 2016/11/25
  5. Poster presentation 大腸菌を用いたSynachocystis sp. PCC6803由来BphA3還元タンパクの探索 第27回日本化学会関東支部茨城地区研究交流会 2016/11/25

Intellectual property rights

  1. Patent 芳香族化合物分解能を有する光合成生物および芳香族化合物の分解方法 特願2010-13334 2010/01/25 特開2011-10651 2011/01/20 特許第5751756号 2015/05/29
  2. Patent 芳香族化合物分解能を有する光合成生物および芳香族化合物の分解方法 特願2009-133311 2009/06/02 特開2011-10651 2011/01/20 特許第5751756号 2015/05/29


  1. 平成23年度科研費審査委員表彰 科研費の第1段審査(書面審査)の検証結果に基づき、模範となる審査意見を付した審査委員。 2011/09/30 H23年度は約5,000名の第1段審査(書面審査)委員の中から49名が選考された。
  2. 日本生化学会 JB論文賞 2004年(第12回) 2004

Alloted class

  1. Biochemistry
  2. Experiments for Bioscience I
  3. Experiments for Applied Chemistry I
  4. Bioscience Seminar
  5. Functional Chemistry Seminar

Memberships of academic societies

  1. The Japanese Biochemical Society
  2. Pritein Science Society of Japan
  3. The Molecular Biology Society of Japan
  4. American Society of Biochemistry and Molecular Biology (ASBMB)

Committee Career

  1. The Japanese Biochemical Society 評議員 2014/10/01-Present
  2. The Japanese Biochemical Society 代議員 2012/10/01-2013/09/30