CHEMICAL SCIENCE

In situ monitoring of the formate dehydrogenase catalytic cycle and its new catalytic mechanism at the monomolecular scale


On March 7, 2023, Beijing time, the team of Professor Fang Baishan, Professor Hong Wenjing and Professor Wang Binju of Xiamen University published a research result entitled “Catalytic cycle of formate dehydrogenase captured by single-molecule conductance” in the journal Nature Catalysis.

By studying the cycling process of formic acid catalyzed by single-molecule coenzyme-dependent formate dehydrogenase, the research team proposed that the NADH that coenzyme-dependent oxidoreductase binds to and binds to its coenzyme is directly converted to NAD+ through in situ hydride transfer reaction, instead of the enzyme catalyzed by the classical Theorel-Chance mechanism, and the coenzyme and enzyme are separated respectively, which is expected to establish a new mechanism of coenzyme-dependent oxidoreductase catalysis.

The co-corresponding authors of the paper are Hong Wenjing, Fang Baishan and Wang Binju; The co-first authors are Zhang Yaohun, Zhuang Xiaoyan, Liu Jia, and Huang Jiacheng.

Schematic diagram of single-molecule conductance monitoring of formate dehydrogenase catalytic reaction and its new catalytic mechanism

Enzymes are a large class of important protein machines that perform catalytic functions in living organisms. They not only provide various reducing forces and energy for cellular metabolic processes, but also receive and transmit signals in the regulation of life activities. There is no doubt that understanding the function and catalytic mechanism of enzymes is an important mission of research in the field of life sciences.

Since 1951, when Hugo Theorell (who won the Nobel Prize in Physiology or Medicine in 1955 for discovering the mechanism of action of oxidases) and Chance (nominated for the Nobel Prize) proposed the famous Theorell-Chance mechanism catalyzed by coenzyme-dependent oxidoreductases, the study of the catalytic mechanism of coenzyme-dependent oxidoreductases has been a hot topic.

However, the ensemble average population study method is still the main research method of enzyme catalytic processes. Due to the lack of characterization methods for systematic analysis of enzyme-catalyzed reaction processes at the single-molecule level, the analysis of enzyme-catalyzed mechanisms through experiments is still a great challenge in bioscience, biotechnology, bioengineering and even life sciences.

Since 1997, Fang Baishan presided over the National Natural Science Foundation of China project “Research on Multi-enzyme Continuous Catalytic Process Based on Charged Membrane and Coenzyme Regeneration”, and has been engaged in the research of coenzyme-dependent oxidoreductases for a long time. Since his surprise discovery in 2001 of the bizarre phenomenon of the oxidation form of the coenzyme catalyzed by glycerol dehydrogenase and the frequent transformation of reduced prototypes, he has questioned the famous Theorell-Chance mechanism and began to focus on its new mechanism; In 2003, he had a whim: to directly replace the enzyme obtained through a series of complex processes such as cell wall breaking with cells containing intracellular oxidoreductase, and was surprised to find that cells without breaking the wall could directly measure the enzyme activity of their intracellular oxidoreductase, thus boldly proposing the idea of a new mechanism: the starting point of coenzyme-dependent oxidoreductase catalysis should be the assembly of oxidoreductases with coenzymes, and the coenzymes after catalytic transformation should be regenerated in situ through the rapid conversion of protons and electrons; Subsequently, the proposed mechanism was supported by innovative design of coenzyme immobilization method and electrochemical method, and the scientific idea of in-situ regeneration of coenzyme in the catalyzed process of coenzyme-dependent oxidoreductase was proposed based on enzyme single molecule to study the enzyme catalytic mechanism. Subsequently, Professor Hong Wenjing, an expert engaged in the assembly and application of scanning tunneling microscopy fracture connection (STM-BJ) instrument, carried out a single-molecule level supporting research on the proposed mechanism, which revealed for the first time the conductivity of formate dehydrogenase binding to coenzyme NAD+. On the basis of a series of previous researches, he applied for the National Natural Science Foundation of China and received funding under the title of “Research on the Conductivity and Catalytic New Mechanism of NAD(P)H-dependent Oxidoreductase”. In the follow-up research, he cooperated with Professor Wang Binju, an expert who is good at chemical calculations, and finally achieved the following gratifying results under the joint research of postdoctoral fellow Zhang Yaohun, Dr. Zhuang Xiaoyan, master’s student Huang Jiacheng and other members of the team under the guidance of Professor Fang Baishan.

The research group used single-molecule scanning tunneling cracking (STM-BJ) technology as a characterization method to successfully establish an enzyme catalytic process research platform based on single-molecule electrical characterization. Taking formate dehydrogenase (FDH) as the research object, the results showed that it showed different conductivity values after binding to reduced or oxidized coenzyme I, respectively. This phenomenon can be used not only to distinguish between different reaction states in the formate dehydrogenase catalytic cycle, but also as a marker for monitoring the formate dehydrogenase catalytic cycle trajectory. Conductance data analysis and statistical results based on artificial intelligence show that the catalytic cycle process of formate dehydrogenase is different from the traditional Theorell-Chance mechanism, and does not undergo a coenzyme dissociation state at the end of its catalysis. Combined with multi-scale simulations, the research team proposed that reduced coenzyme I bound in formate dehydrogenase after the end of the reaction was directly converted to oxidized coenzyme I through in-situ transfer of hydrogen anions, and directly opened a new catalytic cycle. The study shows that fresh NAD+ can easily diffuse near NADH in FDH, resulting in a π-π stacking interaction between the two nicotinamide moieties of NAD+ and NADH. The newly generated NADH can then be converted to NAD+ by in situ hydride transfer reaction with fresh NAD+. Consistent with the experimental results, this calculation shows that the enzyme binding affinity of NADH is much stronger than that of NAD+, suggesting that direct position exchange through the coenzyme dissociation state is very unfavorable.

Figure 1: STM-BJ measures the conductance of a single-molecule FDH junction

Figure 2: Monitoring single-molecule FDH reaction kinetics using the hovering model of STM-BJ

Figure 3:Gibbs free energy was calculated using QM(B3LYP-D3)/MM-MetD

In this work, the dynamic process of enzyme-catalyzed reaction was observed at the single-molecule scale for the first time, and a new mechanism of formate dehydrogenase catalysis at the single-molecule scale was proposed by combining multi-scale theoretical calculations. This study establishes a complete single-molecule enzymatic research platform including single-molecule testing, data analysis and multi-scale simulation, which will open up new ideas for subsequent research on single-molecule enzymatic kinetics, enzyme design, transformation and application. The proposed new mechanism of coenzyme-dependent oxidoreductase catalysis is not only an innovation of Theorell-Chance mechanism, which has been inherited for more than 70 years, but also is expected to update the traditional whole-cell fermentation, multi-enzyme coupling catalysis, enzyme activity detection, biosensor and other technologies, which not only have great scientific significance, but also have broad application prospects. (Source: Science Network)

Related paper information:https://doi.org/10.1038/s41929-023-00928-1



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