Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

Abstract

Pyridine dinucleotides (NAD and NADP) are ubiquitous cofactors involved in hundreds of redox reactions essential for the energy transduction and metabolism in all living cells. In addition, NAD also serves as a substrate for ADP-ribosylation of a number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing regulation, and for cyclic ADP ribose (cADPR)-dependent Ca 2؉ signaling. Pyridine nucleotide adenylyltransferase (PNAT) is an indispensable central enzyme in the NAD biosynthesis pathways catalyzing the condensation of pyridine mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or NaAD). Here we report the identification and structural characterization of a novel human PNAT (hsPNAT-3) that is located in the cytoplasm and mitochondria. Its subcellular localization and tissue distribution are distinct from the previously identified human nuclear PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the catalytic mechanism of the enzyme. The characterization of the cytosolic human PNAT-3 provided compelling evidence that the final steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and mitochondria, potentially contributing to their NAD/NADP pool. The coenzymes NADϩ (H) 1 and NADP ϩ (H) have been known for many decades as the major hydrogen donor or acceptor in hundreds of metabolic redox reactions throughout the cell. Together these nucleotides have a direct impact on virtually every cellular metabolic pathway. Additionally, NAD serves as a substrate for the covalent modification of nuclear proteins by ADP-ribosylation, a process involved in DNA repair and the regulation of genomic instability (1-3). Recently, many new exciting functions have been discovered for this long-known molecule. These include its role as co-substrate in Sir2-mediated histone deacetylation involved in gene silencing regulation and in increasing the lifespan of species ranging from yeast, to worm, to certain mammals (4, 5). Moreover, several derivatives of NAD and NADP were found to be potent intracellular calcium-mobilizing agents and are involved in a variety of Ca 2ϩ -signaling pathways (6 -8). These recent developments brought a significant amount of additional interest to the investigation of cellular NAD biosynthesis and regulation.NMN and/or NaMN adenylyltransferase (NMNAT and/or NaMNAT, collectively named pyridine nucleotide adenylyltransferase, or PNAT) is an indispensable enzyme catalyzing the central step of all NAD biosynthesis pathways (9, 10). It links the AMP moiety of ATP with the nicotinamide mononucleotide (NMN, or its deamidated form NaMN) to form the dinucleotide product NAD (or deamido-NAD, NaAD). A practical aspect of human PNAT function is that it catalyzes the rate-limiting step in the metabolic conversion of the anticancer agent tiazofurin to its active form TAD (tiazofurin adenine dinucleotide, an NAD analog) (11). The development of ti...

NAD and axon degeneration: From the Wlds gene to neurochemistry

“…Each of these adenylyltransferases can participate in both the de novo and salvage NAD + biosynthetic routes. 47,[53][54][55] These three isoforms have distinct cellular localizations. Nmnat-1 is a nuclear protein, while Nmnat-2 and Nmnat-3 are present in Golgi complexes and mitochondria, respectively.…”

NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration

“…However, a DALI search 24 using the structural coordinates of NMNAT1 (Protein Data Bank identity code 1KKU) and NMNAT3 (identity code 1NUR) 25 against the entire Protein Data Bank revealed that both NMNAT1 and NMNAT3 share similarity with chaperone universal stress protein A (UspA; identity code 1JMV) 26 , with a Z score of 5.1, and Hsp100 (identity code 1JBK) 27 , with a Z score of 2.8. A structural superposition of NMNAT1 and UspA showed 13% sequence identity and a root mean squared deviation of 3.0 Å over the entire length of the protein.…”

WldS, Nmnats and axon degeneration-progress in the past two decades

“…The Nmnat enzymes catalyze the last step in three major NAD biosynthesis pathways in both prokaryotes and eukaryotes: the de novo, salvage, and Preiss-Handler-independent pathway (Sasaki et al, 2006(Sasaki et al, , 2009b. Three Nmnat isoforms have been identified in mammals and they differ in subcellular localization and tissue distribution Zhang et al, 2003;Yalowitz et al, 2004). While endogenous Nmnat1 is localized exclusively to nucleus (Schweiger et al, 2001;Zhang et al, 2003), Nmnat2 is mainly expressed in Golgi complex and Nmnat3 is in mitochondria (Berger et al, 2005).…”

“…Three Nmnat isoforms have been identified in mammals and they differ in subcellular localization and tissue distribution Zhang et al, 2003;Yalowitz et al, 2004). While endogenous Nmnat1 is localized exclusively to nucleus (Schweiger et al, 2001;Zhang et al, 2003), Nmnat2 is mainly expressed in Golgi complex and Nmnat3 is in mitochondria (Berger et al, 2005). In terms of tissue distribution, Nmnat1 appears to be almost ubiquitously expressed while Nmnat2 and Nmnat3 exhibit rather specific tissue distributions.…”