Structures of Escherichia coli NAD Synthetase with Substrates and Products Reveal Mechanistic Rearrangements

Abstract

Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.

M. tuberculosis Pantothenate Kinase: Dual Substrate Specificity and Unusual Changes in Ligand Locations

“…A difference in the binding mode of the substrate (nicotinic acid adenine dinucleotide) and the product (nicotinamide adenine dinucleotide) has been observed earlier in the case of NAD synthetase from E. coli. 21 However, the differences in the locations, conformations, and mode of binding observed in the present study are much more extensive.…”

Glutamine-dependent NAD+ Synthetase

“…Addition of NaAD ϩ accelerates the glutaminase activity of all of these mutants 2-to 7-fold but not to the degree observed for wild type (30-fold in specific activity terms). The D593A mutation, which corresponds to an Asp involved in NaAD ϩ binding in both Bacillus subtilis and E. coli NAD ϩ synthetases (Asp-220 and Asp-223, respectively) (17,18), results in an increase of the basal glutaminase activity. Thus, this charge-neutralizing substitution in Saccharomyces cerevisiae Qns1 may cause structural adjustments similar to those induced by NaAD ϩ binding.…”

“…Indeed, we had predicted that the Y532A alteration, which was mapped to the synthetase end of the putative ammonia channel by modeling (Fig. 2) (1), and which corresponds to the nicotinic acid-binding Phe-170 of E. coli NAD ϩ synthetase (18), might eliminate the ability of glutamine-dependent NAD ϩ synthetase to respond to the NaAD ϩ substrate. To determine how specific activity measurements compare with initial rate measurements and to understand the basis for the lethal defect in Y532A,Y601A, we performed a kinetic analysis of the Y601A single and Y532A,Y601A double mutants.…”

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

“…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. deviation of 1.6 Å for 234 equivalent Cα positions in residues 25-272 of draNADS, a Z-score of 30.0, and a sequence identity of 36%) 9 ( Fig.…”

“…[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.…”