Identification, Characterization, and Crystal Structure ofBacillus subtilis Nicotinic Acid Mononucleotide Adenylyltransferase

Abstract

The nadD gene, encoding the enzyme nicotinic acid mononucleotide (NaMN) adenylyltransferase (AT), is essential for the synthesis of NAD and subsequent viability of the cell. The nadD gene in Bacillus subtilis (yqeJ) was identified by sequence homology with other bacterial nadD genes and by biochemical characterization of the gene product. NaMN AT catalyzes the reversible adenylation of both NaMN and the nicotinamide mononucleotide (NMN) but shows specificity for the nicotinate. In contrast to other known NMN ATs, biophysical characterizations reveal it to be a dimer. The NaMN AT crystal structure was determined for both the apo enzyme and product-bound form, to 2.1 and 3.2 Å, respectively. The structures reveal a "functional" dimer conserved in both crystal forms and a monomer fold common to members of the nucleotidyl-transferase ␣/␤ phosphodiesterase superfamily. A structural comparison with family members suggests a new conserved motif (SXXXX(R/K)) at the N terminus of an ␣-helix, which is not part of the shared fold. Interactions of the nicotinic acid with backbone atoms indicate the structural basis for specificity.NAD is an essential molecule in all living cells. In addition to its role in oxidation reduction reactions, in which NAD(H) and its phosphorylated form, NADP(H), act as hydride donors and acceptors, NAD is also important for other cellular processes, such as the activity of NAD-dependent DNA ligases, monoand poly(A)DP-ribosylation of proteins, and production of the intracellular calcium-mobilizing molecules cADPR and NaADP (1, 2).NAD is synthesized via a multi-step de novo pathway or via a pyridine salvage pathway. The enzyme nicotinic acid mononucleotide (NaMN) 1 adenylyltransferase (AT, EC 2.7.7.18) sits at the convergence of these two pathways. NaMN AT catalyzes the conversion of ATP and NaMN to nicotinic acid adenine dinucleotide (NaAD) ( Fig. 1) that is directly processed to NAD by NAD synthetase. The nadD gene, encoding NaMN AT, was the first enzyme demonstrated to be essential for NAD biosynthesis and bacterial cell survival by both the de novo and salvage pathways (3). A number of enzymes demonstrating in vitro adenylyltransferase activity for NaMN and NMN have been identified in eukarya, archaea, and bacteria (4 -11). Along with sequence homology, the specificity of these enzymes for NMN versus NaMN provides a useful method for classifying new genes within this family.Although there is sequence conservation between the eubacterial nadD genes (Fig. 2), sequence alignment of nadD NaMN ATs to the eukaryotic enzymes or archeal enzymes is difficult outside of the region surrounding the (H/T)XGH nucleotidyl transferase consensus sequence. Adenylyltransferases encoded by the nadD gene prefer the nicotinic acid containing NaMN over NMN as a substrate by a factor that ranges from 6:1 to 2000:1 (4, 12, 13). Eubacteria also contain enzymes that demonstrate higher specificity for the nicotinamide-containing NMN. This group includes the products of the nadR gene, which in addition to its regulatory ro...

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

“…The reason for this asymmetry is not clear, but may be related to the different crystal packing environments of the two independent hsP-NAT-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).…”

“…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-Å 2 surface area on each monomer, a second type of dimer interface (e.g.…”

“…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.…”

“…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)(16)(17)(18)(19)(20)(21)(22). In human, two PNAT isoforms (gi 11245478 and gi 12620200) have been cloned and purified, and their kinetic properties were analyzed (23)(24)(25)(26).…”

A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence

“…3). Depletion of NadE furthermore caused NaMN to accumulate, possibly due to product inhibition of NadD or deadenylation of NaAD (28,29). Next, reliance of nonreplicating Mtb on NadE for survival was analyzed during starvation and hypoxia.…”