STRUCTURE AND MECHANISM OF NOCTURNIN AND NADK2: THE DISCOVERY OF A MOLECULAR LINK BETWEEN THE CIRCADIAN CLOCK AND METABOLISM

Abstract

Nicotinamide dinucleotides NADP(H) and NAD(H) are central cofactors driving metabolic reactions in all forms of life. The cellular roles of both NADP(H) and NAD(H) are remarkably well-understood. Nevertheless, here I describe the discovery that the circadian protein, Nocturnin (NOCT), belongs to a new enzyme class, which converts NADP(H) into NAD(H) inside mitochondria. I also describe the use of X-ray crystallography, biochemistry, metabolomics and cell biology to decipher mechanisms of NOCT and its partner kinase, NAD kinase 2 (NADK2).The first part of my thesis work focused on NOCT, which is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, our lab showed that purified NOCT lacks the deadenylase activity. There are 3 major findings in this project: (1) NOCT is not compatible with RNA cleavage; (2) NOCT specifically and directly converts the dinucleotide NADP+ into NAD+, and NADPH into NADH; (3) NOCT is targeted to mitochondria. NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism. The second part of my thesis work focused on NADK2, which phosphorylates NAD+ to maintain the balance between NAD+ and NADP+ in mitochondria. There are 3 major findings in this project: (1) The structure of NADK2 reveals an interlocked loop which stabilizes dimerization. This loop is conserved in NADK2 and absent in the cytosolic NAD kinase, NADK; (2) Dimerization is necessary for the kinase activity of NADK2; (3) Unlike NADK, which forms dimer of dimers upon activation, NADK2 forms spontaneous dimers and exhibits altered cooperativity. These findings reveal divergence between NAD(H)/NADP(H) regulation by cytosolic and bacterial kinases versus mitochondrial kinase, opening a unique new aspect of mitochondrial metabolism.