Author: Zheng Ma

Publisher:

Published: 2015

Total Pages: 179

ISBN-13:

Circadian rhythms are cycles of physiology and behavior that repeat at daily intervals (~24 hours) and are present in all kingdoms of life. The expression of many genes involved in carbohydrate and lipid metabolism is under circadian control. Circadian rhythms are driven by circadian oscillators at the molecular level via transcriptional and post-translational feedback loops generated by a set of interplaying clock proteins. The ratio of reduced to oxidized nicotinamide cofactors has been shown to regulate the DNA binding activity of CLOCK(NPAS2):BMAL1 transcription factor complexes. The structural basis for this interaction is unknown. Crystals were obtained of the bHLH domains of CLOCK and BMAL1 bound to E-box DNA, but the initial crystals failed to produce high-resolution diffraction patterns. The effects and affinities of the nicotinamide cofactors on protein dimer formation or E-box DNA binding were determined, but failed to see any direct evidence for NADPH binding to CLOCK or BMAL1. Nocturnin is a mammalian protein that exhibits a sharp peak of expression at early night, and is thought to regulate circadian gene expression post-transcriptionally by acting as a deadenylase, degrading of poly(A) tails from circadian mRNAs transcribed from clock controlled genes. Genetic studies have shown nocturnin rhythmically regulates target transcripts directly or indirectly in tissues such as adipose, liver, and muscle. Therefore, nocturnin is a potential key mediator between the circadian clock and energy metabolism and a potential target for understanding and treating metabolic disease. The function and mechanism of nocturnin remain unclear. Constructs for producing catalytically active nocturnin have been designed and cloned. Assays for monitoring nocturnin deadenylase activity have been designed and optimized. Substrate scope and preliminary kinetic characterization of nocturnin have been carried out. Recently, three natural products containing a novel core scaffold indolotryptoline with antitumor activity have been isolated from soil actinomycetes. One of them is borregomycin A, of which gene cluster includes two genes encoding proteins with homology to previously described two-component tryptophan halogenases. However, no crystal structures have been reported for the flavin reductase components of two-component halogenases. BorF has been predicted to be its flavin reductase component. The crystal structure of flavin reductase BorF with bound FAD was determined to 2.37 Å in this study.