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

Xuejun ZhangDagger , Oleg V. Kurnasov§, Subramanian KarthikeyanDagger , Nick V. GrishinDagger , Andrei L. Osterman§, and Hong ZhangDagger ||

From the Dagger  Department of Biochemistry and  Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and § Integrated Genomics, Inc., Chicago, Illinois 60612

Received for publication, January 3, 2003, and in revised form, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 Ca2+ 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
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The coenzymes NAD+(H)1 and NADP+(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 Ca2+-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 tiazofurin resistance has been shown to relate mainly to a decrease in PNAT activity (12). Recently, it has also been found that human nuclear PNAT (hsPNAT-1, or NMNAT-1 as in Raffaelli et al. (25)) may play an important role in delaying the Wallerian degeneration in injured axons and synapses: the causative mutation in the slow Wallerian degeneration mouse is found to be a chimeric gene Ube4b/NMNAT encoding hsPNAT-1 fused with the N-terminal region of the ubiquitination factor E4B (13, 14).

Significant progress has been made during the last few years in the enzymological and structural studies of PNATs in various organisms across all three kingdoms of life (15-22). In human, two PNAT isoforms (gi 11245478 and gi 12620200) have been cloned and purified, and their kinetic properties were analyzed (23-26). Human nuclear PNAT-1 was shown to present exclusively in the nuclei and express at high levels in heart, skeletal muscle and, to a lesser extent, in kidney and liver (23). Human PNAT-1 has also been reported to interact specifically with poly(ADP-ribose) polymerase (24, 27) and may be subjected to further regulation by phosphorylation (24). Crystal structures of hsPNAT-1 have been solved in its apo form (20) and in complex with NMN (21), NAD, NaAD, or TAD (19). These structures revealed a hexameric organization of the protein and provided a structural basis for its dual specificity in recognizing both NMN and NaMN substrates (19). Another isoform of human PNAT, hsPNAT-2 (or NMNAT-2 as in Raffaelli et al. (25)) is distinct from the nuclear PNAT-1 (25, 26). It contains 307 residues (versus 279 in hsPNAT-1) and shares 34% sequence identity with the nuclear enzyme. Its tissue expression level is generally low, and it is thought to be primarily expressed in the nervous systems (25). The enzymatic activity of hsPNAT-2 activity is much lower compared with the nuclear form. Both human PNAT isoforms recognize NMN and NaMN substrates equally well; therefore, they are capable of participating in the de novo and salvage/recycling routes of NAD biosynthesis via either NMN or NaMN intermediates.

Historically, PNAT activity has been detected in various animal tissues, including mammary gland, testis, liver, placenta, kidney, thymus, erythrocyte, lymphocyte, and retina (28-33). Although the localization of PNAT activity has been asserted in nuclei (34, 35), a substantial cellular NAD pool is stored in mitochondria (36, 37), and PNAT activity in rat liver mitochondria has also been reported (38). Here we report the identification and characterization of a novel human PNAT, named hsPNAT-3, which is located in the cytosol and mitochondria. Structural analysis of hsPNAT-3 and its complexes with substrates and products provided critical insight in the catalytic mechanism of the adenylyltransfer reaction, which are likely to be shared among all PNATs and some other (T/H)IGH motif-containing adenylyltransferases.

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Materials-- The poly(A)+-RNA filters of 10 human tissues and 10 human cancer cell lines, hybridization buffer, and washing buffer were purchased from Ambion (Austin, TX). The Pfu DNA polymerase was from Stratagene (La Jolla, CA). The Rediprime II DNA labeling kit was from Amersham Biosciences (UK). The pPROEX-1a prokaryotic expression vector was from Invitrogen (Carlsbad, CA). Nickel-nitrilotriacetic acid-agarose was from Qiagen (Valencia, CA). The pEGFP-N1 mammalian expression vector was from Clontech (Palo Alto, CA). The MitoTraker® Red mitochondria-specific fluorescent dye was purchased from Molecular Probes (Eugene, OR). The TEV protease used to remove the His tag of the expressed recombinant proteins was purified in our laboratory from an expression vector kindly provided by Dr. Dave Waugh (NCI, National Institutes of Health, Frederick, MD). The Wizard I and II crystallization screening kits were from Emerald Biostructures (Bainbridge Island, WA).

Expression and Purification of HsPNAT-3-- The hsPNAT-3 cDNA was amplified from a human brain cDNA library (Clontech) and inserted into the pPROEX prokaryotic expression vector (Invitrogen). The resultant pPROEX-hsPNAT3 vector was then transformed into Escherichia coli DH10beta -competent cells (Invitrogen) for protein expression. The cells were induced by adding 0.8 mM isopropyl-1-thio-beta -D-galactopyranoside when grown to A600 = 0.5-0.6 and harvested after shaking at 20 °C overnight. The clarified cell lysate was mixed with the proper amount of nickel-nitrilotriacetic acid-agarose beads (Qiagen) and incubated at 4 °C for 2 h. The bound protein was eluted with 50 mM HEPES (pH 7.2), 300 mM NaCl, and 250 mM imidazole. After the elution, the protein was treated with TEV protease at 4 °C overnight to cleave the His6 tag. As the final purification step, the tag-removed protein was passed through a Mono S ion-exchange column (Amersham Biosciences) and eluted with a gradient of NaCl.

NMNAT- and NaMNAT-coupled Assay-- The hsPNAT-3 activity with both substrates NMN and NaMN was analyzed using a continuous assay coupling NAD formation to alcohol dehydrogenase-catalyzed conversion of NAD to NADH as previously described (39, 40). Briefly, the assay was performed in UV-transparent plastic cuvettes in the six-cuvette autosampler of a DU-640 spectrophotometer (Beckman, Fullerton, CA) at 37 °C. The 500-µl reaction contained 100 mM HEPES-KOH, pH 7.5, 115 mM ethanol, 40 mM semicarbazide, 2 mM ATP, 3 units of alcohol dehydrogenase (Sigma), and ~0.01 µg of hsPNAT-3. The reaction was started by adding 10 µl of 20 mM NMN and monitored at 340 nm over a 20-min period. The NADH extinction coefficient of 6.22 mM-1cm-1 was used for rate calculations. One unit of enzyme was defined as capable of producing 1 µmol of NADH per minute. To measure NaMNAT activity, the procedure was modified by introducing an additional enzymatic step: conversion of NaAD to NAD by NAD synthetase (NADS, EC 6.3.5.1) (40). 0.3 unit of NAD synthetase from Corynebacterium glutamicum (gift from Dr. K. Shatalin, Integrated Genomics Inc.) and 10 mM NH4Cl were added to the same reaction mixture, and the reaction was initiated by adding 10 µl of 20 mM NaMN.

Northern Hybridization-- The DNA template of hsPNAT-3 used for synthesizing a hybridization probe was amplified by PCR from the pPROEX-hsPNAT3 plasmid. About 25 ng of DNA was used for probe synthesis in the presence of [32P]CTP using the Rediprime II system (Amersham Biosciences). The synthesized probe was purified with a Micro Bio-spin 30 column (Bio-Rad) to remove the non-incorporated nucleotides. The two poly(A)+-RNA filters (the normal cell multiple tissue blot and multiple cancer cell line blot from Ambion) were prehybridized at 42 °C for 30 min before being hybridized with the 32P-labeled probe at 42 °C for 16 h. After hybridization, the filters were washed according to the manufacturer's instruction. The autoradiography was set at -70 °C for 7 days. The blots were subsequently washed and rehybridized with radiolabeled beta -actin probe to normalize for mRNA loading levels.

Mammalian Cell Culture and GFP Localization-- HEK293 (human kidney epithelial) and HeLa cells (kind gift from Dr. Helen Yin, UT Southwestern Medical Center at Dallas, TX) were cultured in the Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum (Harlan Bioproducts for Science). The cells were incubated at 37 °C with 5% CO2. To make the C-terminal GFP fusion mammalian expression constructs for hsPNAT-1, hsPNAT-2, and hsPNAT-3, the cDNA of each of the three genes was amplified by PCR from their respective prokaryotic expression plasmids and inserted into the pEGFP-N1 mammalian expression vector (Clontech). The resultant vectors pEGFP-hsPNAT1, pEGFP-hsPNAT2, and pEGFP-hsPNAT3 were transfected into cells using SuperFect reagent (Qiagen) when the cell density reached about 70% confluence. The localization of the expressed GFP fusion proteins was examined after 2-day transfection with a Zeiss Axiovert 35 fluorescence microscope. The co-localization of hsPNAT-3 and mitochondria-specific dye was observed under a Zeiss LSM 510 confocal fluorescence microscope.

Crystallization and Data Collection-- The hsPNAT-3 crystals were obtained using the hanging drop, vapor diffusion method. 1.5 µl of hsPNAT-3 (20 mg/ml) was mixed with 1.5 µl of reservoir solution containing 100 mM sodium cacodylate, pH 6.0-7.0, 200 mM Li2SO4, and 20-25% polyethylene glycol 400, and equilibrated against the reservoir. The crystals of the complexes of hsPNAT-3 with various substrates and products, including NMN, AMP-CPP, NAD, NaAD, and a ternary complex with both NMN and AMP-CPP, were obtained by co-crystallization. All crystals were directly frozen in liquid propane, and the diffraction data were collected on a RAXIS-IV image plate detector equipped with a Rigaku-H3R x-ray generator and Osmic mirrors at a temperature of 100 K. The data were processed with the HKL2000 package (41), and the space group was later determined to be P43212 with cell dimensions a = b = 79.36 Å, and c = 146.2 Å. There are two hsPNAT-3 monomers in the asymmetric unit. The crystal data of all six crystals are listed in Table I.

Structure Determination and Refinement-- The structure of the apo hsPNAT-3 was determined by the molecular replacement method in CNS (42) using the coordinates of the nuclear hsPNAT-1 monomer as the initial search model. The cross-rotation search found two solutions with correlation coefficients (c.c.) of 0.0686 and 0.0556, respectively, which were significantly higher than the rest of the solutions (the next highest peak had a c.c. of 0.0325). After translation search and rigid body refinement with the two monomers located, the c.c. was 0.647 and the R-factor was 0.39. Subsequent refinement and model building were then carried out using the programs CNS (42) and O (43). The refined apo-hsPNAT-3 structure was used as the initial model for the refinement of the complex structures. Various ligands and solvent molecules were included in the models according to the difference densities at later stages of the refinement. The refinement statistics of all six structures are listed in Table I.

    RESULTS AND DISCUSSION
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A BLAST search (44) of the non-redundant protein sequence data base (NCBI, National Institutes of Health, Bethesda, MD) using hsPNAT-1 (gi 11245478) as query found several human homologous proteins that also possesses the signature (T/H)IGH and ISSTXXR adenylyltransferase motifs (18, 45), suggesting that these proteins may also encode for PNAT (see Supplemental Material). In particular, a 252-amino acid human protein sequence (gi 14029540), termed here hsPNAT-3, shares 50% identity with hsPNAT-1 and 82% identity with a mouse protein (gi 13543122) that presumably is the mouse ortholog of hsPNAT-3. We have cloned and overexpressed hsPNAT-3 and experimentally confirmed that indeed it has the predicted adenylyltransferase activity. Like the two other isoforms, hsPNAT-3 has comparable activity with both physiological substrates (5.6 units/mg for NaMN and 4.7 units/mg for NMN at saturating concentration of either substrate). Both activities of hsPNAT-3 are lower compared with hsPNAT-1 (38 and 30 units/mg, respectively), but being ~10-fold higher compared with hsPNAT-2. Together with the previously reported hsPNAT-1 and hsPNAT-2, three human PNAT isoenzymes are now identified. To understand the cellular and physiological roles of these different human PNATs, we studied the tissue distributions and subcellular localization of the three different hsPNATs by Northern hybridization and expression of GFP fusion proteins in cultured kidney epithelial HEK293 and HeLa cells.

Northern Blot Analysis of hsPNAT-3-- The overall mRNA levels of hsPNAT-3 in normal tissues and cancer cell lines were low compared with hsPNAT-1 (23) (Fig. 1). Clear hybridization bands of a 1.1-kb transcript were detected in mRNA blots of human lung and spleen, and weaker reaction bands were detected in placenta and kidney. There was almost no hybridization signal detected in all the cancer cell lines tested (data not shown). This tissue distribution pattern is different from both human PNAT-1 and PNAT-2 (23, 25). There appears to be no overlapping in tissue distributions between the three hsPNAT isoforms, suggesting that expression of these isoenzymes are differentially controlled at transcription level and each may have a distinct cellular and physiological role.


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Fig. 1.   Expression of hsPNAT-3 in human tissues. The poly(A)+-RNA from human tissues were analyzed by Northern hybridization with a 32P-labeled probe corresponding to the coding region of hsPNAT-3. The blot was subsequently washed and rehybridized with radiolabeled beta -actin to normalize for mRNA loading levels.

Subcellular Localization of the Three Human PNAT Isoenzymes-- We monitored subcellular localization of the three human PNAT isoenzymes tagged with GFP and overexpressed in HEK293 and HeLa cells (Fig. 2). We found that GFP-hsPNAT1 is located exclusively in the nuclei (data not shown), consistent with the results from earlier immunofluorescent studies (24). GFP-hsPNAT3 is shown to exist outside nucleus. Further examination of the co-localization of GFP-hsPNAT3 with the red mitochondria-specific dye showed that hsPNAT-3 is also present in mitochondria (Fig. 2A). GFP-hsPNAT2, on the other hand, is found to have a very specific localization outside nucleus that does not overlap with the mitochondria-specific dye (Fig. 2B). Its exact location in the cell remains to be determined.


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Fig. 2.   Subcellular compartmentalization of hsPNAT-2 and hsPNAT-3. A, HEK293 and HeLa cells transfected with plasmid pEGFP-hsPNAT3 were stained with MitoTracker® Red and examined under a confocal fluorescence microscope. The three images show the same group of cells visualized using different color channels. B, HeLa cells transfected with pEGFP-hsPNAT2 and stained with MitoTracker® Red. The distribution of the fusion protein GFP-hsPNAT2 in HEK293 cells is the same as in the HeLa cells and is not shown.

The results obtained here demonstrate for the first time that two human PNAT isoenzymes are expressed outside cell nucleus. Whereas the highly compartmentalized localization of hsPNAT-2 remains to be determined, hsPNAT-3 is shown to express in the cytosol and mitochondria. Together with previous observations that a substantial cellular NAD pool is stored in mitochondria (36, 37) and that PNAT activity has previously been detected in rat liver mitochondria (38), the identification of a cytosolic and mitochondria human PNAT-3 gives further evidence that at least part of the NAD biosynthesis pathways exists in human cytosol and mitochondria.

The Tetrameric Organization of hsPNAT-3-- To learn the structural mechanisms of the function of hsPNAT-3, we have determined three-dimensional structures of hsPNAT-3 in its apo form and in complexes with each substrate (ATP analog AMP-CPP or NMN), product (NAD or NaAD), and with both NMN and AMP-CPP. The overall structure of the hsPNAT-3 monomer is very similar to the nuclear hsPNAT-1, with root mean square deviation (r.m.s.d.) between the superimposed Calpha atoms about 0.9 Å (Fig. 3A). The structure contains a central parallel six-stranded beta  sheet surrounded by helices in a typical Rossmann fold topology. A region consisting of residues 106-125 is disordered. The corresponding region in hsPNAT-1 contains the nuclear localization signal (NLS) sequence and is also disordered. The main differences between the structures of the two hsPNAT isoforms are located at the loop regions between residues 52 and 57, between residues 166 and 171, and between residues 192 and 199 preceding the ISSTXXR motif (Fig. 3A).


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Fig. 3.   The overall structure of hsPNAT-3. A, stereo view of the Calpha trace of hsPNAT-3 monomer (red) superimposed with hsPNAT-1 (blue). Every 20th residue is labeled. B, stereo diagrams of hsPNAT-3 tetramer. Each monomer is colored differently. The disordered regions are shown as dotted loops. Two orthogonal views are shown. The bound NAD molecules are shown as ball-and-stick representations.

The active site residue arrangement in hsPNAT-3 is also very similar to that in hsPNAT-1. However, the higher order oligomerization states of the two hsPNAT isoforms are different. There are two independent hsPNAT-3 monomers in the asymmetric unit of the crystal. Inspection of the crystal packing indicated that hsPNAT-3 forms a tetramer (Fig. 3B), which is consistent with the deduced molecular weight from gel filtration chromatography. The hsPNAT-3 tetramer is tightly packed, and the four monomers in the tetramer are related by a D2 symmetry. One of the dimer interfaces (e.g. between the yellow and orange monomers in Fig. 3B) is similar to that observed in hsPNAT-1 (19-21). A similar dimer interface is also conserved in Bacillus subtilis NaMNAT, which exists as dimers in solution (18). Although this commonly shared dimerization interface buries a 1223-Å2 surface area on each monomer, a second type of dimer interface (e.g. between the yellow and cyan monomers in Fig. 3B) that is unique for hsPNAT-3 buries about the same amount of surface area (1244 Å2). Both types of interface are largely polar in nature. Similar to hsPNAT-1, hsPNAT-3 is also highly positively charged with a theoretical pI of 9.28, but these charges are rather dispersed and no surface charge clustering is observed.

Ligand Binding-induced Conformational Changes-- Compared with the apo-hsPNAT-3, all the substrate/product-bound hsPNAT-3 complexes display some common trends in the ligand binding-induced conformational changes (Fig. 4). The r.m.s.d. values between Calpha atoms of the apo and various ligand-bound complexes range from 0.47 to 0.56 Å, whereas differences between ligand-bound structures are smaller, ranging from 0.24 to 0.39 Å. The structures of the NAD- and NaAD-bound complexes are nearly identical with an r.m.s.d. of 0.18 Å.


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Fig. 4.   Stereo diagram of superposition of the active site between apo-form (in yellow) and NAD-bound (in cyan) hsPNAT-3. The bound NAD (in orange) is shown in ball-and-stick representation. Several protein residues discussed in the text are also shown. This and subsequent figures were generated with MOLSCRIPT (53) and BOBSCRIPT (54) and rendered with gl_render (L. Esser, unpublished program).

The ligand binding-induced conformational changes near the NMN binding site are local and mostly in the residues that make contact with NMN (Fig. 4). Upon binding NMN or NAD/NaAD, residues 50-56 and 144-149 around the NMN binding site made various degrees of adjustment (usually less than 1 Å) and in general closed in on the bound substrate so that the contacts with the ligand are optimized. Similar ligand binding-induced movements around the NMN binding site were also observed in hsPNAT-1 (19, 20).

At the adenine binding site, however, local side-chain movements as well as large main-chain conformational changes in loop 193-199 occurred upon binding the adenine containing ligands (Fig. 4). In the apo-hsPNAT-1 structure, side chains of Met-21, Met-25, and Arg-167 are pointed toward the adenine binding site and would have clashed with a bound adenylate ligand (Fig. 4). Val-196 located on the loop 193-199 is also close to the adenine binding site. Upon binding the ATP substrate or NAD/NaAD, the Arg-167 side chain is now stacked against adenine ring, and the side chains of the two methionines, Met-21 and Met-25, are rotated away. The loop 193-199 underwent large conformational changes with the Val-196 side chain now moved outward by ~6 Å (Fig. 4). The new conformation of loop 193-199 enables a hydrogen bond to be formed between N6 of adenine and the main chain carbonyl of Asn-198 (see below).

It needs to be pointed out that such adenine nucleotide binding-induced conformational changes in loop 193-199 were not observed in the monomer B in the asymmetric unit. The conformation of this loop in monomer B in the apo structure is similar to that of the ligand-bound form, and no drastic conformational change occurs upon adenylate binding. The reason for this asymmetry is not clear, but may be related to the different crystal packing environments of the two independent hsPNAT-3 monomers. The relatively small ligand binding-induced conformational changes in both hsPNAT-1 and hsPNAT-3 are in contrast to the drastic conformational changes observed in the bacterial (E. coli and B. subtilis) NaMNATs, which occur at both ATP and NMN binding sites (17, 18).

The ATP Binding Site-- The bound AMP-CPP (a non-hydrolyzable ATP analog) in the binary and ternary complexes adopts essentially identical conformations and makes the same interactions with the enzyme. The conformation of the AMP portion of AMP-CPP is also very similar to that of the AMP moiety of the bound NAD. The detailed interactions between bound AMP-CPP and surrounding protein atoms are shown in Fig. 5A. In particular, the ATP triphosphate tail makes extensive interactions with the enzyme. The alpha -phosphate interacts with the main-chain amide groups of Gly-14 and Ser-15. The beta -phosphate interacts with Nepsilon 2 of the His-22 side-chain imidazole. This histidine residue is the invariant second histidine in the (H/T)IGH motif that has been shown to be critical for the catalysis (16). The gamma -phosphate of the ATP interacts with the side chains of Lys-56, Arg-206, and Thr-203, with the main-chain amide of Thr-203, and with the Lys-140 side chain from monomer B. Thr-203 and Arg-206 are part of the ISSTXXR motif located at the N terminus of an alpha  helix and are highly conserved in many (T/H)IGH motif-containing nucleotidyltransferases (18). Our structures, along with the ATP-bound structures of Methanococcus jannaschii NMNAT (15) and E. coli phosphopantetheine adenylyltransferase (PPAT) (46), showed that this ISSTXXR motif is involved in the binding of the gamma -phosphate of the bound ATP and is likely to be in the position to bind the leaving pyrophosphate group as well.


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Fig. 5.   The ATP and NMN binding sites in hsPNAT-3. A, stereo view of the ATP binding site. B, the Fo - Fc omit map for NMN molecule in monomer B of the NMN binary complex showing the alternative conformations of NMN phosphate. The map is contoured at 2sigma . C, stereo view of the NMN binding site. The substrates ATP and NMN and a bound sulfate molecule are shown as bonds colored according to atom types. The two alternative conformations of the bound NMN are shown. Relevant protein residues are shown in ball-and-stick representation. The hydrogen bonds are indicated by dotted lines.

In the hsPNAT-3 complexes with NAD or NMN, a sulfate molecule was located near the bound ligand. Superposition with the ATP complex shows that this sulfate molecule occupies a position near the gamma -phosphate of ATP and forms extensive interactions with the surrounding protein atoms. Such a sulfate is also observed in the Methanobacterium thermoautotrophicum NMNAT·NAD complex structure (16). We have not observed a Mg2+ ion in any of the hsPNAT-3 complex structures, because no Mg2+ is present in the crystallization buffer. It is likely that a Mg2+ ion is needed for the catalysis (see below) but is not required for substrate binding.

The NMN Binding Site-- In the hsPNAT-3 and NMN binary complex, alternative conformations exist in the phosphate portion of NMN in monomer B (Fig. 5B). In one of the two conformers, the phosphate is bent toward the pyridine ring, whereas in the second conformer the phosphate is in a more extended conformation. The distance between the phosphor atoms in the two conformers is 2.7 Å. The NMN bound to monomer A adopts exclusively the "bent" conformation. The reason for this asymmetric binding of NMN in the two hsPNAT-3 monomers of the asymmetric unit is not clear. In monomer A where NMN adopts the bent conformation, the side chain of Tyr-53 rotates about 20° compared with that in the monomer B to avoid potential close contact with the NMN phosphate. As a result, the side-chain hydroxyl of Tyr-53 moves 1.2 Å and is now hydrogen-bonded to the NMN phosphate oxygen (2.53 Å) (Fig. 5C). Otherwise, there are few interactions between protein atoms and NMN phosphate in the bent conformation. In the extended conformation, the NMN phosphate interacts with the main-chain amide and side-chain hydroxyl of Ser-14. It also interacts with the Phe-15 main-chain amide through a water molecule (Fig. 5C). Such extended conformation is also observed in the NMN and NaMN binary complexes of hsPNAT-1,2 and in the closely related PPAT binary complex with phosphopantetheine (46). It appears that the extended NMN conformation forms more interactions with the protein and may be energetically more favorable. It was thus somewhat unexpected that the NMN in the hsPNAT3·NMN binary complex adopted the bent conformation in the absence of ATP. Notably, a sulfate molecule is present in the NMN binary complex that partially overlaps with the binding site of the ATP gamma -phosphate (Fig. 5C). The presence of this sulfate might contribute to keep the NMN in the bent conformation.

The conformation of the nicotinamide ribose portion of the bound NMN is essentially the same as the corresponding region in NAD. The detailed interactions between NMN and protein is similar to that in hsPNAT-1 and is shown in Fig. 5C. In particular, the pyridine ring stacks against the Trp-148 side chain and forms a face-to-edge interaction with Trp-90. The exocyclic carboxyamide group interacts specifically with the main-chain amide of Thr-93 and main-chain carbonyl of Leu-147. The Leu-147 side chain also forms van der Waals contacts with the pyridine ring. Two highly conserved active site water molecules (w1 and w2) that are seen in hsPNAT-1 and bacterial NMNATs are also conserved in hsPNAT-3 and interact with the carboxyamide moiety of nicotinamide. One of these water molecules (w1) is linked to the amide group of the nicotinamide, main-chain amide of Lys-149, and the side-chain Ndelta 1 of His-152. As discussed below, this water molecule may play a role in the dual specificity of the enzyme.

Structural Basis for the Dual Substrate Specificity of hsPNAT-3-- To understand the structural basis for the dual substrate specificity of hsPNAT-3, we have solved the structures of hsPNAT-3 complexed with either NAD or NaAD. As mentioned before, the pyridine ribose portion of the dinucleotide occupies the same binding site as that in the NMN binary complex. The interactions between the nicotinamide moiety and surrounding protein atoms as well as the two active site water molecules are very similar to that observed in the hsPNAT-1 (19) (Fig. 5C). Previously, we have found that in hsPNAT-1·NaAD complex, besides the subtle local conformational changes, an active site water molecule also moved toward the carboxylate, partially compensating the negative charge of NaAD (19). In the hsPNAT3·NaAD complex, however, no active site water movement is observed that could provide additional interactions with the negatively charged carboxylate. Upon close inspection of the surrounding protein groups, we found that one of the active site water molecules (w1) that interacts with the carboxylate of NaAD is hydrogen-bonded to His-152 (Fig. 5C). We hypothesize that this water molecule may interact with the amide group of NAD as a hydrogen acceptor and change to a hydrogen donor when interacting with the carboxylate oxygen of NaAD, partially neutralizing the negative charge of NaAD. Coordination to a histidine side chain should enable this water molecule to perform this dual functional role.

The NMN·AMP-CPP Ternary Complex Structure and the Mechanism of Adenylyltransfer Reaction-- In the hsPNAT-3 ternary substrate complex structure, the conformation of the NMN molecule adopts the bent conformation similar to that in the NMN binary complex. Steric hindrance due to the presence of the ATP triphosphate tail appears to prevent NMN from adopting an extended conformation when both substrates are bound (Fig. 6A). The AMP-CPP conformation is essentially the same as that of the AMP-CPP in the binary complex. Compared with the respective binary complexes, the B-factors are much higher for both substrates in the ternary complex (Table I), indicating that simultaneously bound substrates in the enzyme active site are less stable and may be of partial occupancies. Additionally, the average B-factor of protein atoms in the ternary complex is also somewhat higher than that of the binary complexes, suggesting that the enzyme might become more flexible upon binding of both substrates.


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Fig. 6.   Comparison of conformations of the bound ligands in hsPNAT3. A, the electron density of NMN and AMP-CPP in hsPNAT-3 ternary substrate complex. The Fo - Fc omit map is contoured at 2sigma . Here the NMN molecule is shown to adopt exclusively the bent conformation. B, superposition of ligands in the NMN binary complex (yellow, both NMN conformers are shown), in the AMP-CPP·NMN ternary complex (cyan), and in the NAD product complex (orange). The putative Mg2+ ion binding site (as inferred from the M. jannaschii NMNAT·ATP complex structure) is indicated by a purple sphere.


                              
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Table I
Crystal data and refinement statistics of hsPNAT-3 crystals

Notably, in the NMN and NaMN binary complexes obtained for hsPNAT-12 and in the binary complex of the related E. coli PPAT with substrate phosphopantetheine (46), the phosphorylated adenylyl acceptors all adopt the extended conformation that overlaps the corresponding portion of the product. However, in the hsPNAT-3 ternary substrate complex, the presence of the ATP triphosphate tail makes the extended NMN phosphate conformation impossible, and as a result the NMN phosphate has to adopt a highly bent conformation. Because there are few interactions with the enzyme that would stabilize the NMN phosphate in its bent conformation, we assume that this conformation is less favorable or less stable than the "extended" conformation. The alternative bent and extended NMN conformations in the PNAT active site provided critical insight into the catalytic mechanism of PNAT, which has been proposed to use an in-line nucleophilic attack of the NMN phosphate on the ATP alpha -phosphate (47). The propensity of NMN to adopt an extended conformation would bring the NMN phosphate close to the ATP alpha -phosphate for such an attack. The interactions of the enzyme with ATP and NMN phosphates would help to stabilize the two negatively charged phosphate groups in close proximity. Although the required Mg2+ is not observed in our crystal structures, its position may be inferred from the M. jannaschii NMNAT·ATP complex structure, in which a Mg2+ is found to be coordinated to the oxygens from all three ATP phosphates (15) (Fig. 6B). The location of the Mg2+ between the ATP alpha -phosphate and the NMN phosphate suggests that it is likely to play a role in mediating the nucleophilic attack between the two phosphate groups. There are extensive interactions between the enzyme and the ATP gamma -phosphate but few interactions with the beta -phosphate of ATP (Fig. 5A). This binding mode would not only help to make the beta ,gamma -pyrophosphate a good leaving group but also favor its rearrangement after the cleavage of the alpha /beta -phosphodiester bond.

The structure of hsPNAT-3 complexed with both AMP-CPP and NMN described here is the first ternary substrate complex structure reported for any (T/H)IGH motif-containing adenylyltransferase. The reaction mechanism proposed here is likely to be shared among other PNATs and some adenylyltransferases as well. It should be noted that the NMN molecule in the hsPNAT3·NMN binary complex can also adopt the bent conformation, indicating that the energetic difference between the two conformations (bent versus extended) is marginal. It is likely that several factors, including the tendency of NMN to adopt an extended conformation, Mg2+ coordination, and interactions with the surrounding protein atoms, all contribute synergistically to facilitate the adenylyltransfer reaction.

Three Distinct hsPNAT Isoforms and NAD Metabolism in Human-- The PNAT activity has been detected in a variety of animal tissues, including mammary gland, testis, liver, placenta, thymus, kidney, retina, lymphocyte, and erythrocyte (28-33). Many reports have shown that this activity resides in the nuclei (34, 35). However, substantial cellular NAD pool is stored in mitochondria (36, 37, 48), and PNAT activity has been reported from the rat liver mitochondria as well (38). The first human PNAT characterized at the molecular and atomic structural level contains a typical monopartate nuclear localization signal sequence (NLS) and was shown to exist exclusively in nuclei by the immunofluorescent studies (24) and by the localization of the expressed GFP fusion protein (this study). This human nuclear PNAT (hsPNAT-1) is highly expressed in heart and skeletal muscles and weakly expressed in kidney, pancreas, and liver (23). The second human PNAT isoform, hsPNAT-2, does not possess a typical NLS and has been predicted to be cytosolic (25). Here we have shown that its localization outside nuclei is highly compartmentalized and that it is not expressed in mitochondria. The mRNA level of hsPNAT-2 is low compared with the nuclear hsPNAT-1 and exists mostly in the nervous system (25). The third human hsPNAT-3 reported here exists in cytosol and mitochondria, but not nucleus. Its tissue distribution is distinct from hsPNAT-1 and hsPNAT-2 and is present in lung and spleen and, to a lesser extent, in placenta and kidney, where no expression of the other two hsPNAT isoforms were detected. All three hsPNAT isoenzymes are expressed in cancer cells at much lower levels, which are assumed to be close to the critical level for cell survival (29, 49). Consequently, PNAT has been regarded as a potential target for anticancer chemotherapy, because the minimal inhibitory concentration lethal to cancer cells is not likely to cause serious damage to normal cells (49).

Human PNAT-1 and -3 have close orthologs in mouse and probably in other mammals as well (see Supplemental Material). They share 82% sequence identity to their respective mouse orthologs. So far no mouse orthologs has been found for hsPNAT-2, which may be due to the fact that the sequencing of mouse genome is yet to be completed.

Currently most of the enzymatic steps involved in human NAD/NADP biosynthesis are understood, and several relevant pathways have been asserted. Genes encoding all of the steps in the five-stepped de novo biosynthesis of quinolinate from tryptophan and the subsequent synthesis of NAD and NADP from quinolinate have been characterized either in human or in mouse (9, 10, 50-52). Several NAD salvage routes in human have also been deduced from comparative genome analysis in combination with existing biochemical data (26). One of these routes, the salvage of nicotinamide ribose, is directly related to the hypothesized mechanism of tiazofurin activation (11, 19). This mechanism includes a concerted action of ribosylnicotinamide kinase to produce NMN from nicotinamide ribose, and PNAT to convert NMN directly to NAD. So far NAD degradation and recycling pathways are still poorly understood. Two major types of NAD hydrolytic degradation are known: the NAD-pyrophosphohydrolase type (producing NMN and AMP) and the NAD-glycohydrolase type (producing nicotinamide and ADP-ribose) (50, 51). NMN-specific activity of PNAT may be involved in the recycling of NMN formed by the first mechanism. The second hydrolytic reaction is directly coupled to the non-redox functions of NAD(P), such as ADP-ribosylation, histone deacetylation, and cyclic ADP ribose-dependent Ca2+ signaling. Nicotinamide formed in the course of these processes can be recycled via the salvage pathways.

Relatively little is known about tissue distribution, intracellular compartmentalization, and regulation of these multiple components of NAD metabolism in human cells, which complicates accurate inference of biologically relevant NAD biosynthetic routes. Clearly PNAT plays a central role in all of these pathways. In contrast to an early dogma, the existence of a cytosolic and mitochondria hsPNAT-3 strongly suggests that final steps of NAD biosynthesis may occur outside of the nucleus. All of the three characterized human PNAT isoforms display dual substrate specificity toward either NMN or NaMN substrate and thus are flexible to participate in either de novo or salvage/recycling pathways via either intermediate. The nuclear hsPNAT-1 is undoubtedly involved in the NAD regeneration in the nucleus, but its role in NAD de novo biosynthesis is less obvious. It is tempting to speculate that hsPNAT-3 may be one of the key participants in NAD biogenesis outside of the nucleus. Further experiments are required to revisit the early dogma on the cellular NAD biosynthesis, which was based solely on the localization of the major PNAT activity inside nuclei.

Because the NAD molecule serves both as redox cofactor in many basic cellular metabolic pathways and as substrate for several regulatory reactions, understanding the NAD biosynthesis and how it is regulated may be important for deciphering the link between metabolism and various regulatory events, such as gene silencing, Ca2+ signaling, and protein ADP-ribosylation. As the central enzyme in NAD metabolism, human PNATs appear to be subject to regulations by phosphorylation and interaction with ADP-ribosyltransferase (24). The identification and characterization of three distinct human PNAT isoenzymes represent a step forward in human NAD biosynthesis studies. Further experiments in various types of cells and in a variety of conditions are required to characterize the respective roles of these PNAT isoenzymes in a complex network of NAD metabolism and regulation.

    ACKNOWLEDGEMENTS

We thank Leeju Wu in Steve McKnight's laboratory for help with the Northern blot analysis, Susan Sun in Helen Yin's laboratory for help with the use of fluorescence microscopes, Yingming Zhao for help with the mammalian cell culture, Hongtao Yu for helpful discussions, and Lisa Kinch for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM65243 (to H. Z.) and The Robert A. Welch Foundation Grant I-5051 (to N. V. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Fig. S1 and its legend on one text page.

The atomic coordinates and the structure factors (code 1NUP, 1NUQ, 1NUR, 1NUS, 1NUT, and 1NUU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

|| To whom correspondence should be addressed. Tel.: 214-648-9299; Fax: 214-648-9099; E-mail: zhang@chop.swmed.edu.

Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M300073200

2 H. Zhang, unpublished results.

    ABBREVIATIONS

The abbreviations used are: NAD, nicotinamide adenine dinucleotide; NaAD, nicotinic acid adenine dinucleotide; NMN, nicotinamide mononucleotide; NaMN, nicotinic acid mononucleotide; NMNAT, NMN adenylyltransferase; NaMNT, NaMN adenylyltransferase; PNAT, pyridine nucleotide adenylyltransferase; AMP-CPP, adenosine 5'-(alpha ,beta -methylene)triphosphate; PPAT, phosphopantetheine adenylyltransferase; r.m.s.d., root mean square deviation; GFP, green fluorescent protein; EGFP, enhanced GFP; NLS, nuclear localization signal; c.c., correlation coefficient.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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