領 域 名 生命機能科学
教員氏名 櫻 庭 春 彦
研究分野 酵素工学
研究キーワード：超好熱菌，アーキア，耐熱性酵素，X線結晶構造解析
最近の研究課題
１．構造情報に基づく新規D-アミノ酸脱水素酵素の創製
メゾジアミノピメリン酸脱水素酵素（DAPDH）は、D-アミノ酸のNAD(P)依存性の脱アミノ反応を可逆的に触媒する唯一の脱水素酵素として知られており、各種オキソ酸とアンモニアからのD-アミノ酸 1 段階合成への利用が期待される。D-アミノ酸は各種医薬品や農薬の原料として有用であり、近年その需要が高まっているが、DAPDHを用いればこれを簡便に合成することが可能になる。しかしDAPDHは不安定なものが多く、また基質特異性が高くDAPに特異的に作用するため、その応用展開が阻まれてきた。我々は最近、好熱菌由来の安定なDAPDHの構造解析に成功し、基質結合に関与するアミノ酸残基を推定した。さらに部位特異的変異により、基質特異性の決定に重要な役割を持つ残基の同定を試みた。その結果、D94Aの 1 アミノ酸変異により、基質特異性が劇的に変化することがわかり、フェニルピルビン酸に高い反応性を示す新規酵素の創製に成功した。この変異酵素は医薬品原料として有用なD-フェニルアラニンの 1 段階合成への利用が期待される。
２．超好熱アーキア由来脱水素酵素が示す特異的な補酵素結合様式
メチオニン、スレオニン、イソロイシンは微生物や植物にのみ存在する代謝経路よって、アスパラギン酸から生合成される。この経路に存在するホモセリン脱水素酵素（HseDH）は、ヒトには存在しないため、新規抗菌剤設計のための分子標的として注目される。我々は、超好熱アーキアに見出したHseDHについて構造解析と部位特異的変異実験を行い、NADPの強力な結合がNAD(P)依存性脱水素酵素の触媒活性にとって妨げとなるという、補酵素特異性決定の新しいメカニズムを明らかにした。さらに我々は、超好熱菌由来 sn-グリセロール-1-リン酸脱水素酵素にも新たな補酵素結合様式を見出しており、脱水素酵素のNAD/NADP特異性を決定するメカニズムの多様性を提唱している。 （cover image ⇒）
代表的な研究業績
- Hayashi, J., et al. (2017) Structure-based engineering of an artificially generated NADP-dependent D-amino acid dehydrogenase. Applied and Environmental Microbiology, in press.
- Hayashi, J., et al. (2017) Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri. Acta Crystallographica, D73, 428-437 .
- Wakamatsu, T., et al. (2016) Structural insights into L-tryptophan dehydrogenase from a photoautotrophic cyanobacterium Nostocpunctiforme. Applied and Environmental Microbiology, in press.
- Hayashi, J., et al. (2016) Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculumcalidifontis. Proteins, 84, 1786-1796 .（本研究はcover image に採択された）
- Ohshida, T., et al. (2016) First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.Protein Expression and Purification, 126, 62-68 .
- Hayashi, J., et al. (2015) Crystal structures of a hyperthermophilic archaeal homoserine dehydrogenase suggest a novel cofactor binding mode for oxidoreductases. Scientific Reports, 5, 11674.
- Sakuraba, H., et al. (2012) Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix. The Journal of Biological Chemistry, 287, 20070-20080.
Research Area: Life Science & Biotechnology
Research Specialization : Enzyme Engineering
Name: SAKURABA, Haruhiko
Keywords: hyperthermophiles, archaea, thermostable enzyme,x-ray crystal structure analysis
Recent Research
1. Structure-based creation of a novel D-amino acid dehydrogenase
meso-Diaminopimelate dehydrogenase (DAPDH) acts stereoselectively on the D-center of substrate. Thus, the enzyme is expected to be useful for one-step production of D-amino acids, which are often utilized as source materials for industrial production of medicines and agrochemicals. However, its high substrate specificity for DAP and instability have proved to be the major disadvantage for the practical application of DAPDH. Recently, we determined the crystal structure of thermostable DAPDH and estimated the amino acid residues related to substrate binding. We used site-directed mutagenesis to identify the residues responsible for determining the enzyme’s substrate specificity and found that a single mutation (D94A) caused a striking change in the substrate specificity of the enzyme. The mutant exhibited extremely high activity for reductive amination of phenylpyruvate. This is a major advantage of using the D94A mutant for one-step synthesis of D-phenylalanine.
2. Unique coenzyme binding mode of hyperthermophilicdehydrogenase
Homoserine dehydrogenase (HseDH) is a key enzyme in the biosynthetic pathway for the synthesis of methionine, threonine, and isoleucine in plants and microorganisms. HseDH is thought to be a potential target for the structure-based design of antibiotics, as the enzyme is not present in mammals. We recently determined the crystal structure of HseDH from the hyperthermophile and found that this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity. More recently, we observed another variation on a structural basis for cofactor preference using hyper-thermophilic sn-glycerol-1-phosphate dehydrogenase. Our studies indicate that the molecular basis for the cofactor preference in NAD(P)-dependent dehydrogenases may have much more variation than expected
代表的な研究業績
- Hayashi, J., et al. (2017) Structure-based engineering of an artificially generated NADP-dependent D-amino acid dehydrogenase. Applied and Environmental Microbiology, in press.
- Hayashi, J., et al. (2017) Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri. Acta Crystallographica, D73, 428-437 .
- Wakamatsu, T., et al. (2016) Structural insights into L-tryptophan dehydrogenase from a photoautotrophic cyanobacterium Nostocpunctiforme. Applied and Environmental Microbiology, in press.
- Hayashi, J., et al. (2016) Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculumcalidifontis. Proteins, 84, 1786-1796 .（本研究はcover image に採択された）
- Ohshida, T., et al. (2016) First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.Protein Expression and Purification, 126, 62-68 .
- Hayashi, J., et al. (2015) Crystal structures of a hyperthermophilic archaeal homoserine dehydrogenase suggest a novel cofactor binding mode for oxidoreductases. Scientific Reports, 5, 11674.
- Sakuraba, H., et al. (2012) Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix. The Journal of Biological Chemistry, 287, 20070-20080.
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