Abstract
Nicotinamide mononucleotide adenylyltransferase (NMNAT) is the central enzyme of the NAD biosynthetic pathway. Three human NMNAT isoforms have recently been identified, but isoform-specific functions are presently unknown, although a tissue-specific role has been suggested. Analyses of the subcellular localization confirmed NMNAT1 to be a nuclear protein, whereas NMNAT2 and -3 were localized to the Golgi complex and the mitochondria, respectively. This differential subcellular localization points to an organelle-specific, nonredundant function of each of the three proteins. Comparison of the kinetic properties showed that particularly NMNAT3 exhibits a high tolerance toward substrate modifications. Moreover, as opposed to preferred NAD+ synthesis by NMNAT1, the other two isoforms could also form NADH directly from the reduced nicotinamide mononucleotide, supporting a hitherto unknown pathway of NAD generation. A variety of physiological intermediates was tested and exerted only minor influence on the catalytic activities of the NMNATs. However, gallotannin was found to be a potent inhibitor, thereby compromising its use as a specific inhibitor of poly-ADP-ribose glycohydrolase. The presence of substrate-specific and independent nuclear, mitochondrial, and Golgi-specific NAD biosynthetic pathways is opposed to the assumption of a general cellular NAD pool. Their existence appears to be consistent with important compartment-specific functions rather than to reflect simple functional redundance.
Highlights
For many years the pyridine nucleotides have been recognized as vital to all organisms because of their essential role in energy transduction
Endogenous NMNAT1 was localized exclusively to the nucleus [12], NMNAT2- and NMNAT3-GFP fusion proteins were targeted to the cytosol, NMNAT3 was partially found within the mitochondria [27]
To conduct a detailed functional characterization of the three different human NMNAT isozymes, their cDNAs were cloned into the pQE30 prokaryotic expression vector, and the respective proteins were overexpressed in E. coli
Summary
CDNA Cloning for Prokaryotic Expression and Purification of Recombinant Human NMNATs—Human NMNAT1 was cloned into the pQE30 prokaryotic expression vector (Qiagen) as described previously [12]. The purified human recombinant NMNATs were incubated with the indicated substrates in reaction buffer (50 mM Tris HCl, pH 8, 5 mM MgCl2, 200 mM NaCl). Relative substrate specificities of human recombinant NMNATs were measured by incubation for 1 h at room temperature with 1 mM mononucleotide and 2 mM purine nucleotide when measuring the transfer activity or 500 M dinucleotide and 5 mM pyrophosphate when measuring the dinucleotide cleavage activity. Eukaryotic Expression of Recombinant Human NMNATs—The cDNAs encoding human NMNAT1 and NMNAT2 were cloned into the pFLAG-CMV-4TM eukaryotic expression vector (Sigma) providing the recombinant proteins with an N-terminal FLAG tag. NMNAT3 was cloned into the pFLAG-CMV-5TM eukaryotic expression vector (Sigma) providing the recombinant protein with a C-terminal FLAG tag. Mitochondria were visualized by Mitotracker (Molecular Probes/Invitrogen) according to the manufacturer’s protocol
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