The Cabinet Office recommends the ideal society for Japan to aim for in the near future.Society 5.0”, the large amount of information received from sensors is analyzed as big data and returned to people in various forms Especially in recent years, when interest in health care has become size, real-time monitoring of biological materials and multi-sensing have attracted attention. Among them, electrochemical biosensors based on a conjugated system of enzyme reaction and electrode reaction have been practically used as blood sugar level sensors because they can detect target substances with high selectivity under mild conditions, in particular, ” “direct electron transfer (DET)” reactions, in which an enzyme transfers electrons directly to an electrode, are simple reaction systems consisting of only an enzyme and an electrode, and are therefore environmentally compatible and have a high degree of design freedom. However, only about 30 enzymes are capable of reporting this reaction, and only a very small number of substances can be measured.
We aim to create new DET-type enzymes by using fructose dehydrogenase (FDH), which has a strong DET-type catalytic activity, as a model enzyme. This enzyme is a catalytically active subunit that has a flavin adenine dinucleotide (FAD) as the catalytic site.cConsists of an electron transport subunit that hasheterotrimer(Figure 1). Because of its unique activity, FDH has been extensively studied from both a fundamental and an applied perspective. In addition, we succeeded in structural analysis using cryo-electron microscopy in 2022, making it possible to hold discussions based on information about the complete structure of the enzyme. Therefore, we investigated the detailed mechanism of the catalytic reaction of FDH from the perspective of structural biology and computer science.
Figure 1. Schematic diagram of the three-dimensional structure and electron transfer pathway of FDH
2-1 Identifying amino acid residues involved in the catalytic reaction of FDH using computer science, structural analysis, and biochemistry.
As a result of docking simulations and homology searches, threeAmino acid residues are mutatedIt has been suggested that (N1146, H1147, N1190) is important for enzyme substrate recognition and catalytic reactions. Therefore,Site-directed mutagenesisWe created mutants in which these amino acid residues were replaced with different ones. As a result of identifying these mutants using electrochemical measurements and structural analysis using a cryo-electron microscope (using one from the JEOL YOKOGUSHI Cooperative Research Institute, Graduate School of Frontier Biology , Osaka University), we found that the mutant enzymes observed changes in substrate affinity and enzyme activity. Figure 2(A) summarizes the positions of amino acid residues we focused on. Since the mutation to N1146 reduced the affinity for the original substrate fructose while maintaining the enzymatic activity, we expected a change in specificity for another substrate. As a result of determining reactivity with other sugars, it was determined that the mutant (N1146Q) in which N1146 was replaced by a glutamine residue (Q) had better reactivity with tagatose, which has a similar structure to fructose (Figure 2 (B )).
Figure 2. (A) Elements involved in the catalytic reaction of FDH, (B) Reactivity with tagatose
2-2 Explaining and predicting substrate selection using stochastic models
The three-dimensional structures of the newly generated mutants were analyzed by cryo-electron microscopy, and docking simulations were performed. Figure 3 (A) is an example of the simulation results, and we were able to identify the hydrogen bond needed to identify fructose. In addition, as shown in Figure 3(B), according to the theoretical formula, the docking score (DS) is determined by the Michaelis constant (Kshowed a good correlation with the logarithm of M). This means that substrate selectivity can be predicted from the three-dimensional structure of the enzyme. On the other hand, we also verified whether the computer-generated predicted structure could reproduce the actual analytical results. As a result, as shown by the red dotted line in Figure 3(B), the result was different from the calculation using cryo-electron microscopy structure, indicating that there is still room for improvement in the computer based structure prediction, which has made amazing progress in recent years I understand.
Figure 3. (A) FDH docking simulation, (B) correlation between DS and Michaelis constant
This research is the first report in the world that elucidates the catalytic mechanism of FDH through the combination of protein computational science, structural analysis, and biochemistry, as well as the development of a third-generation synthetic biosensor using FDH as a model enzyme that will be the academic foundation that will result. In the future, based on the information obtained this time, we will combine computer-based machine learning and reaction prediction methods to significantly change the substrate specificity of the catalytic active site and analyze materials biological and useful compounds with a high sensitivity platform that can sense with high efficiency. Therefore, this research not only has a high academic value, but also accelerates the development of catalysts through interdisciplinary collaboration and the social implementation of third generation biosensors, contributing to the result of a sustainable society that is friendly to the environment and living organisms.
タイトル:Structural and electrochemical elucidation of biocatalytic mechanisms in direct electron transfer type D-fructose dehydrogenase
(Elucidation of the catalytic mechanism for fructose dehydrogenase direct electron transfer using structural and electrochemical methods)
Author: Eole Fukawa, Yohei Suzuki, Taiki Adachi, Tomoko Miyata, Fumiaki Makino, Hideaki Tanaka, Keiichi Namba, Keisei Sowa, Yuki Kitazumi, Osamu Shirai
Issue: Book of electrochemistry490, 144271 (2024), DOI: 10.1016/j.electacta.2024.144271.
This research was supported by Japan Agency for Medical Research and Development AMED BINDS system (JP22ama121003), Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (JP21H01961, JP22K14831), JST Innovative GX Technology Creation Project (GteX) (JPMJGX23B4 ), Kyoto This event was supported by donations to the university (Mr. Hiroo Kaku, Mr. Wang Holong, and Mr. Yasunori Hamano).
