Structural adaptation of an interacting non-native C-terminal helical extension revealed in the crystal structure of NAD⁺synthetase fromBacillus anthracis

Abstract

The crystal structures of NH(3)-dependent NAD+ synthetase from Bacillus anthracis as the apoenzyme (1.9 A), in complex with the natural catalytic products AMP and pyrophosphate (2.4 A) and in complex with the substrate analog adenosine 5'-(alpha,beta-methylene)triphosphate (2.0 A) have been determined. NAD+ synthetase catalyzes the last step in the biosynthesis of the vitally important cofactor NAD+. In comparison to other NAD+ synthetase crystal structures, the C-terminal His-tagged end of the apoenzyme adopts a novel helical conformation, causing significant compensatory changes in the region. The structural accommodations observed in B. anthracis NAD+ synthetase are remarkable in the absence of adverse affects on enzyme activity. They also illustrate a rare example of the influence of a non-native C-terminal His-tag extension on the structure of a native protein. In contrast to the apoenzyme, when AMP and pyrophosphate or adenosine 5'-(alpha,beta-methylene)triphosphate are bound, the C-terminus adopts a conformation that allows ATP binding and overall the structure then resembles other NAD+ synthetase structures. The structures of NAD+ synthetase complexes from B. anthracis are compared with published X-ray crystal structures of the enzyme from B. subtilis, Escherichia coli and Helicobacter pylori. These comparisons support the novel observation that P1 and P2 loop ordering is not a consequence of crystal contacts but rather a consequence of intrinsic intramolecular interactions within the ordered subunit.

Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

“…A comparison with a recently published 3D structure of baNadE (1.28-Å average C ␣ RMSD) reveals a similar dimer organization (Fig. 2 A), in which each subunit contributes to a partially shared active site (28). Most of the active site residues that provide key interactions with ATP, Mg ions, and ammonia (as originally mapped in the most detailed structure-function analysis of bsNadE (31)) are conserved in ftNadE*.…”

“…Hence, it was tempting to suggest the product of the F. tularensis nadE gene a candidate for the proposed functional role of NMN synthetase (termed ftNadE*). Despite the straightforward chemical analogy, this alternative activity was previously never detected (20,21), and, in most cases, this activity was not assayed for any of the characterized members of the NadE family (22)(23)(24)(25)(26)(27)(28). To test the bioinformatic predictions described above, we performed the kinetic and structural characterization of purified recombinant ftNadE* enzyme.…”

Structural Analysis of the NH3-dependent NAD+Synthetase from Deinococcus radiodurans

“…deviation of 1.0 Å for 267 equivalent Cα positions in residues 11-280 of draNADS, a Z-score of 39.8, and a sequence identity of 58%), 5 (ii) banNADS (PDB code 2PZ8, r.m.s. deviation of 1.1 Å for 269 equivalent Cα positions in residues 10-281 of draNADS, a Z-score of 39.6, and a sequence identity of 59%), 7 (iii) ecoNADS (PDB code 1WXI, r.m.s. deviation of 1.1 Å for 258 equivalent Cα positions in residues 11-281 of draNADS, a Z-score of 36.7, and a sequence identity of 59%), 6 and (iv) ftuNADS (PDB code 3FIU, r.m.s.…”

“…[5][6][7][8][9] As the prokaryotic and eukaryotic NADS differ in size, enzymatic activity and substrate requirements, NADS is an attractive target for the development of a new class of antibiotics. The NADS homolog (UniProt code Q9RYV5) in Deinococcus radiodurans encodes a protein of 287 amino acid residues, with 59% sequence identity to that of E. coli.…”

NAD(P) Biosynthesis Enzymes as Potential Targets for Selective Drug Design

“…glutaminedependent. Enzymes of the first class have been typically found in most bacteria, like E. coli [142], B. subtilis [139], S. typhimurium [143], H. pylori [144], and B. anthracis [145], while the second group is reported in eukaryotes [146,147] and selected prokaryotes like M. tuberculosis [148,149], and Synechocystis sp [76]. Both classes display a highly conserved C-terminal domain, responsible for the NADS activity, including a P-loop motif characteristic of the N-type family of ATP pyrophosphatases [150].…”