【公開日：2025.06.10】【最終更新日：2025.05.01】
課題データ / Project Data
課題番号 / Project Issue Number
24NM0079
利用課題名 / Title
NAD結合型NAD依存性脱水素酵素、 ニコチノプロテインにおける特徴的なループ構造の役割の解明
利用した実施機関 / Support Institute
物質・材料研究機構 / NIMS
機関外・機関内の利用 / External or Internal Use
外部利用/External Use
技術領域 / Technology Area
【横断技術領域 / Cross-Technology Area】（主 / Main）物質・材料合成プロセス/Molecule & Material Synthesis（副 / Sub）-
【重要技術領域 / Important Technology Area】（主 / Main）次世代バイオマテリアル/Next-generation biomaterials（副 / Sub）-
キーワード / Keywords
Sensor chip NTA, Protein-Protein binding affinity,バイオセンサ/ Biosensor
利用者と利用形態 / User and Support Type
利用者名（課題申請者）/ User Name (Project Applicant)
薛 厚丞
所属名 / Affiliation
東京農工大学 工学府応用生命工学専攻_大学院
共同利用者氏名 / Names of Collaborators in Other Institutes Than Hub and Spoke Institutes
ARIM実施機関支援担当者 / Names of Collaborators in The Hub and Spoke Institutes
利用形態 / Support Type
（主 / Main）機器利用/Equipment Utilization（副 / Sub）-
利用した主な設備 / Equipment Used in This Project
報告書データ / Report
概要（目的・用途・実施内容）/ Abstract (Aim, Use Applications and Contents)
Nicotinoprotein is a unique group of NAD+ dependent dehydrogenases (NADDh) that bind to NAD+ tightly and catalyze the reaction without using free NAD+. This research aims to study the role of the unique insertion loop from nicotinoprotein. The insertion loop from SDR (Short-chain Dehydrogenase/Reductase) nicotinoprotein ReCADh, was introduced to the typical SDR AfBHBDh and created 4 mutants. Insertion loop deleted, ReCADh mutant Δ39-49 CADh also created. Furthermore, an in silico study was also performed. The AfBHBDh mutants did not show dehydrogenase activity in the presence of electron acceptor, DCIP, without free NAD+, but the Km, KD value and binding affinity between NAD+ and enzyme was increased compared to AfBHBDh. Δ39-49 CADh showed the ability lost bind with NAD+ tightly and was unable to utilize free NAD+. From these results, the insertion loop interferes the entry NAD+ and creates a more energy favorable binding environment for NAD+, but the loop is only a necessary condition and couldn’t achieve NAD+ binding only relying on loop introduction. This study deepened the understanding of the NAD+ binding mechanism of SDR and demonstrates potential applications in enzyme engineering and metabolic engineering. However, other studies of potential factors of NAD+ tight binding of SDR are anticipated.
実験 / Experimental
Method of SPR measurementSPR measurement was performed with a Biacore X100 and enzyme were immobilized on a sensor chip NTA at 25 ̊C. The running buffer consisted of 20 mM P.P.B. (pH 7.0), 150 mM NaCl, and 0.5% Tween 20 was used. The sensor chip was washed by 350 mM EDTA in running buffer for 60 sec. Then 0.5 mM of NiCl2 was injected for 60 sec and washed by 3 mM EDTA. 30 μg/ml of enzyme was injected to be immobilized onto the NTA-chip in each cycle by multi-run mode automatically.In the association phase, injection of NAD+ solution of 0.01, 0.1, 0.5, 1, 2, 3 mM started at 0 sec and stopped at 60 sec. In the dissociation phase, sensorchip was washed by running buffer for 60 sec. Finally, the sensor chip was regenerated by running buffer containing 350 mM EDTA.
結果と考察 / Results and Discussion
KD value of AfBHBDh
for NAD+
was 1.3×10-6 M and BHBDh36-48 Cins for NAD+ was 8.7×10-6 M. For AfBHBDh and BHBDh36-48 Cins, the
reliable kon and koff were not determined
because the sensorgram changed just after 0 sec and 60 sec so rapidly to
determine kon and koff . The SPR analysis revealed that the KD value of BHBDh36-48 Cins was 6-fold higher than that of AfBHBDh, indicating that BHBDh36-48 Cins has a lower affinity than AfBHBDh. This result was similar to the NAD+ concentration-dependent assay in that the Km value increased between AfBHBDh and BHBDh36-48 Cins. Combining the results of Km and KD values, it was considered that the insertion loop plays a role in interfering with the entry and exit of NAD+ from BHBDh36-48 Cins and SDR nicotinoprotein. This is beneficial due to the characteristic of nicotinoprotein that prevents the entry and exit of external NAD+.
図・表・数式 / Figures, Tables and Equations
図1
その他・特記事項（参考文献・謝辞等） / Remarks(References and Acknowledgements)
成果発表・成果利用 / Publication and Patents
論文・プロシーディング（DOIのあるもの） / DOI (Publication and Proceedings)
口頭発表、ポスター発表および、その他の論文 / Oral Presentations etc.
特許 / Patents
特許出願件数 / Number of Patent Applications：0件
特許登録件数 / Number of Registered Patents：0件