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Determining if Circadian Clock-Control of Sorbitol Dehydrogenase Generates Rhythms in L-Iiditol Levels and Impacts the NAD+/NADH Ratio in Neurospora crassa
Abstract
The endogenous circadian clock controls daily rhythms in gene expression, metabolism, and behavior [1-3]. Circadian clock control of specific metabolites has been studied in different organisms; however, little is known about the clock regulation of metabolites in fungi, including the clock model organism Neurospora crassa. Mass spectrometry of metabolites isolated from WT and clock mutant N. crassa cells at different times of the day identified ~2000 metabolites, and the levels of 8 of the annotated metabolites appeared to be controlled by the circadian clock. One clock-controlled metabolite of interest was L-iditol because the production of L-iditol can alter the levels of the critical metabolite NAD+ . In a reversible reaction, Liditol 2-dehydrogenase, also known as sorbitol dehydrogenase (SDH), catalyzes the production of L-iditol from L-sorbose and reduces NADH to NAD+ . These data suggested that clock control of this pathway would lead to daily rhythms in the levels of NAD/NADH, which may in turn drive rhythms in metabolism. Two SDH genes are encoded in the N. crassa genome. Independent validation of clock control of L-iditol levels, and the dependence of L-iditol rhythms on SDH-1 and/or SDH-2 are being examined using metabolomics. To determine if the clock regulates SDH-1 and/or SDH-2 protein levels, SDH-1 and SDH-2 are tagged with an epitope tag and protein levels will be analyzed from cultures harvested at different times of the day. SDH-1, but not SDH-2, was shown to be necessary for normal SDH enzyme activity, and deletion of SDH-1 and SDH-2 altered the ratio of NAD+ /NADH. Experiments are underway to determine if clock control of the L-iditol pathway leads to rhythms in the levels of NAD+ / NADH.
Citation
Taylor, Cody A (2023). Determining if Circadian Clock-Control of Sorbitol Dehydrogenase Generates Rhythms in L-Iiditol Levels and Impacts the NAD+/NADH Ratio in Neurospora crassa. Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /199888.