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October 30, 2002 Structure of Human Nicotinamide/Nicotinic Acid Mononucleotide Adenylyltransferase (NMNAT)T. Zhou1, D.R. Tomchick1, D.D. Binns1, N.V. Grishin1, H. Zhang1, O.V. Kurnasov2, A.L. Osterman2, and V.E. Marquez3 Nicotinamide adenine dinucleotide (NAD) is a coenzyme (nonprotein part of an enzyme) involved in many metabolic reactions inside the cell, as well as DNA repair and calcium signaling. NAD results from the addition of adenylate (one of RNA’s building blocks) to the molecule nicotinamide mononucleotide (NMN), a process catalyzed by the enzyme NMN adenylyltransferase (NMNAT). The enzyme is also involved in activating the anticancer agent tiazofurin. To understand better the role of NMNAT in NAD biosynthesis and tiazofurin conversion, scientists have used x-rays produced at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory and the Advanced Photon Source at Argonne National Laboratory to determine the structure of NMNAT with different ligands, providing insight into the molecular mechanisms of the enzyme’s active site.
Scientists have recently found that NAD is a substrate or a co-factor in the SIR2-like histone deacetylase, responsible for gene silencing and the increase of lifespan of many species, including yeast, worm, and mammals. Also, several derivatives of NAD are intracellular calcium mobilizing agents in various calcium signaling pathways. NAD results from the addition of adenylate (one of RNA’s building blocks) to the molecule nicotinamide mononucleotide (NMN), a process catalyzed by the enzyme NMN adenylyltransferase (NMNAT). NAD can also be synthesized by the following two successive processes: addition of adenylate to the molecule nicotinate mononucleotide (NaMN), leading to nicotinate adenine dinucleotide (NaAD), and addition of an amide group (organic compound containing the CONH2 radical) to NaAD, leading to NAD. The first process is catalyzed by NMNAT and the second by NAD synthetase. (A synthetase is an enzyme that catalyzes the union of two molecules.) The processes are illustrated at: http://hhmi.swmed.edu/Labs/hz/nad1.htm Human NMNAT also catalyzes part of the metabolic conversion of the anti-cancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD), an NAD analogue. The development of tiazofurin resistance has been shown to relate mainly to a decrease in NMNAT activity. The process is illustrated at: http://hhmi.swmed.edu/Labs/hz/tiazofurin.htm
Human NMNAT, located within the cell nucleus, recognizes both NMN and NaMN substrates. To understand the enzymatic properties of NMNAT and how its activity is regulated, we have solved the crystal structures of human NMNAT attached to several ligands, including NAD, NaAD, and TAD, to 2.2-angstrom (Å) resolution. The data were produced and collected by using x-rays generated at beam line X12B of the National Synchrotron Light Source at Brookhaven National Laboratory and beam line 19ID of the Advanced Photon Source at Argonne National Laboratory in Illinois. Our results show that NMNAT bind to NMN and NaMN with high affinity. In particular, a water molecule in the active site appears to play a critical role in the recognition of NaMN (right part of figure). By looking at the structure of NMNAT attached to TAD, we found that TAD molecule adopts a similar conformation as the native ligand NAD, and that TAD and NAD form essentially the same interactions with NMNAT. Additional functional groups on tiazofurin molecule also may enable more favorable interactions between tiazofurin and NMNAT. Further kinetic measurement will be needed to fully characterize the molecular interactions between human NMNAT and tiazofurin. The results presented here represent a first step in our effort to decipher the metabolic pathways of NAD biosynthesis and how it is regulated in humans. We have recently identified an additional cytosolic form (inside the cell cytoplasm instead of the cell nucleus) of human NMNAT, its structural determination being well underway. BEAMLINE FUNDING PUBLICATION FOR MORE INFORMATION |
The National Synchrotron Light Source at Brookhaven National Laboratory in New York, is a national user research
facility funded by the U.S. Department of Energy's Office of Basic Energy Science. The NSLS operates two electron
storage rings: an X-Ray ring and a Vacuum UltraViolet (VUV) ring which provide intense light spanning the electromagnetic
spectrum from the infrared through x-rays. Each year over 2300 scientists from universities, industries and government
labs perform research at the NSLS.
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Nicotinamide adenine dinucleotide (NAD) is a coenzyme (nonprotein
part of an enzyme) that has been known for decades as the major
hydrogen donor or acceptor in many metabolic reactions inside the
cell, as well as the modification of nuclear proteins by ADP
ribosylation, a process involved in DNA repair and the regulation of
genomic instability. 