Figure 1.
Scheme of the two-step reaction catalyzed by NAD synthetase.
Table 1.
Genomic arrangement of the synthetase and glutaminase components in NAD synthetase and other families of ATP-dependent amidotransferases.
Figure 2.
Genomic arrangements of functionally coupled glutaminase (GAT) and synthetase (NADS) components.
Comparative genome analysis revealed 4 different genomic arrangements of GAT and NADS components: a) two domain organization (fusion); b) physical clustering; c) remote occurrence; d) absence of glutaminase. Their relative distribution across Archaea, Bacteria, and Eukaryotes is also shown (left side).
Figure 3.
Biochemical characterization of T. thermophilus glutaminase and NAD synthetase subunits.
SDS-page analysis of Ni-NTA affinity column (A) and gel filtration chromatography (B) elution fractions show that His-tagged recombinant T. thermophilus NADS and untagged GAT tend to co-purify. (C) Kinetic characterization of T. thermophilus S-subunit and G/S complex.
Figure 4.
Analysis of Nit11 and SK51 S. typhimurium mutant strains.
(A) Schematic of S. typhimurium nadE mutant strains. Nit11 mutant features a missense mutation at nucleotide 143 yielding the amino acid substitution AN. SK51 mutant features a single nucleotide deletion (G at position – 51 considering as +1 the first base of the start codon) that is just upstream of the -35 box regulatory region of the promoter. Predicted regulatory sequences are indicated in the bottom line. (B) Relative expression level of nadE in the wild type and in the two classes of mutants nit11 (S48N) and SK51 in four different growth conditions: rich medium (1), minimal medium supplemented with 20 mM NH3 (2), MM supplemented with 5 mM (3) or 20 mM (4) glutamine. (C) Kinetic characterization of wild type and S48N S. typhimurium NAD synthetase. Initial rates were measured by spectrophotometrical coupled (SPEC) assays. The kinetic parameters Km and kcat are apparent values determined at fixed (saturating) concentrations of co-substrates. For fixed substrates, concentrations were: 2 mM ATP, 2 mM NaAD, and 40 mM NH3. Errors represent standard deviation.
Figure 5.
Phylogenetic analysis of NAD synthetase enzyme family.
(A) Schematic representation of NAD synthetase phylogenetic tree (full version is in Figure S1) constructed based on synthetase domain. Defined types of NAD synthetase genes – “Fused” (type F), “Clustered” (type C), “Remote” (type R) and “None” (type N) are highlighted by red, green, cyan and magenta colors, respectively. The whole tree is partitioned by topology into clusters, which are designated as I–VII branches. (B) Schematic representation of species tree with mapping of NAD synthetase gene types (full version is in Figure S2). Genomes containing single NAD synthetase gene of F, N, C, and R types are depicted by red, green, cyan and magenta colors, respectively. Genomes that possess more than one NAD synthetase gene are divided into “multiple F”, “multiple N”, “single F – single N” and “all others” genome groups, which are highlighted by dark red, dark green, orange and yellow colors, respectively.
Figure 6.
Contact regions between synthetase (blue) and glutaminase (cyan) domains in NADS from Mycobacterium tuberculosis.
The majority of interacting residues were found in following structural regions-the α9, α18 helices and the extended C-terminal loop, which are highlighted by pink, green and yellow colors, respectively.
Table 2.
Distribution of two-component glutamine-utilizing NADS signature elements across phylogenetic branches.
Figure 7.
Tentative evolutionary scenario of one- and two-domain form of NAD synthetase enzyme family.