In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light–dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.

No abstract

In photosynthetic organisms, it is recognized that the intracellular NADP+/NADPH ratio is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADP+/NADPH ratio quantitatively estimated to date. In the present study, the light-response of the NADP+/NADPH ratio was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light-response of NADP(H) observed using PCI extraction was qualitatively consistent with the NADPH fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light-response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition, indicating that some NADP(H) is not light-responsive. Specifically, 64% of total NADP(H) was observed as non-light-responsive in the wild-type cells. The variation of the intracellular NADP+/NADPH ratio is limited to a narrow range due to the presence of non-light-responsive NADP(H).

Biological assimilation of CO2 to produce sugars occurs in metabolic cycles with an autocatalytic nature, such as the Calvin cycle and reverse citric acid cycle. The formose reaction, in which sugars are non-enzymatically synthesized from formaldehyde under basic conditions, involves such an autocatalytic cycle and has attracted much interest from the viewpoint of the abiotic chemical synthesis of sugars. However, many side reactions are indiscriminately accelerated by hydroxide ions, which results in a very low selectivity of sugar formation. Here we report non-enzymatic sugar synthesis under neutral conditions using mono-oxometalate as a catalyst to form an autocatalytic cycle. The construction of an autocatalytic reaction system under neutral conditions significantly improved the selectivity of sugar formation. It was also demonstrated that abiotically synthesized sugars could sustain the growth of microbial cells.

Biological assimilation of CO2 to produce sugars occurs in metabolic cycles with an autocatalytic nature, such as the Calvin cycle and reverse citric acid cycle. The formose reaction, in which sugars are non-enzymatically synthesized from formaldehyde in alkaline solutions, involves such an autocatalytic cycle and has attracted much interest from the viewpoint of the abiotic chemical synthesis of sugars. However, many side reactions are indiscriminately accelerated by hydroxide ions in an alkaline aqueous solution, which results in a very low selectivity of sugar formation. Here we report non-enzymatic sugar synthesis in a neutral aqueous solution using mono-oxometalate as a catalyst to form an autocatalytic cycle. The construction of an autocatalytic reaction system in a neutral aqueous solution significantly improved the selectivity of sugar formation. It was also demonstrated that abiotically synthesized sugars could sustain the growth of microbial cells.