The evolution and new exploration of the catalytic mechanism of oxidoreductase

Oxidoreductases play an important role in the metabolism of life so that revealing their catalytic mechanism is momentous for further reveal the influence of electron and proton transfer on the growth of living organisms. More importantly, oxidoreductase accounts for about one-third among all enzymes newly registered on the BRENDA website, of which half using NAD(P)H/NAD(P)+ as a coenzyme. Hugo Theorell is awarded a Nobel Prize in 1955 for achievements that discovered the nature and mode of action of oxidation enzymes in 1951. Cooperate with Britton Chance, they propose the famous Theorell-Chance mechanism based on the research of the catalysis kinetics mechanism of liver alcohol dehydrogenase, which has always been a hot topic since then. Thus, in the past 70 years, the study of the mechanism and its modification has always been a focus of researches since the classic oxidoreductase’s catalysis mechanism is put forward in 1951. Based on the previous work, we review the evolution of the catalytic mechanism of oxidoreductases systematically and thus put forward two valuable scientific problems. (1) Do intracellular coenzymes bound to oxidoreductases enzymes have to dissociate after oxidation or reduction? (2) Do intracellular NAD(P)H-dependent oxidoreductases enzyme and its corresponding coenzyme have self-assembly? Then, we also detailed introduce some discoveries of oxidoreductases catalytic mechanism based on our recent work. Based on the whole-cell catalytic process of 1,3-propanediol oxidoreductase and glycerol dehydrogenase, we find that the whole-cell can also catalyze the extracellularly NAD+ to NADH. After then, technology development brings us advanced precise instruments, which give us a deeper insight into the catalyzation process at a single-molecular level. Among them, the STM-BJ (scanning tunneling microscope break junction) technique provides excellent performance in spatial resolution and electrical detection sensitivity, which has been one of the most frequently used techniques in single-molecule electrical detection in recent years. Thus, we provide the first demonstration of the STM-BJ technique for investigating charge transport through a single active enzyme junction, in which the binding of NAD+ with proteins boosts the charge transport by over 2100% than neutral FDH. Combine with site-specific mutagenesis, we demonstrate the conductance of FDH-NAD+ highly correlate with their bioactivities. And we will also employ it to study the real-time conductivity of single NAD(P)H-depending formate dehydrogenase in the catalytic process to reveal the new catalytic mechanism at the single-molecule level in future work. Studying the combination of single NAD(P)H-depending oxidoreductase and its co-enzyme and the reaction of substrate catalyzing is an original innovative research, which will help us to understand the essence of enzyme catalyzation in life activity and to improve technologies in directed enzyme evolution and development of biomedical drugs. The proposed catalytic mechanism of the NAD(P)H-depending oxidoreductase is expected not only to revolutionize the theory that has been followed for 70 years but also to improve a wide range of traditional technologies, such as whole-cell fermentation, multiple enzymes coupling catalyzation, enzyme activity detection, bio-sensor and so on.