Absorbance at 280 nm ( mAU )

1. Fig. S1 A 180 100 Absorbanceat 280 nm (mAU) 20 15 10 Elution volume (ml) MW (kDa) 6.5 7 13.5 14.5 15 15.5 16 16.5 18.5 19.5 B Fig. S1. Gel filtration profile of HsNadE2. A) Elution profile of HsNadE2 on a Superose 6 column, the elution volume corresponds to 85 kDa. B) SDS-PAGE analysis of gel filtration chromatography fractions.

2. Fig. S2 A B D C F E Fig. S2– Determination of kinetic parameters for NAD synthetases from A. brasilense and H. seropedicae. Initial velocity (Vo) for the substrates ammonium (A,C and E) and glutamine (B, D and F). Average data from triplicate experiments ± SD.

3. Fig. S3 MW (kDa) 1 2 3 4 5 6 92 67 43 30 Fig. S3- SDS-PAGE analysisofA. brasilense NadE1Gln, NadE2GlnandNadENH3.MW molecular markers. His-tag proteins: AbNadE1Gln-His (Lane 1), AbNadE2Gln-His (Lane 3), AbNadE3NH3-His (Lane 5). Taglessproteins used in this work: AbNadE1Gln (Lane 2), AbNadE2Gln (Lane 4), and AbNadE3NH3 (Lane 6). ). The gel wascoomassiebluestained. Thelineindicatesthesplicing in theimageofthesame gel.

4. Fig. S4 A 180 100 Absorbanceat 280 nm (mAU) 10 15 20 Elution volume (ml) MW (kDa) 13.5 14 14.5 15 15.5 16 17 19.5 B Fig. S4– Gel filtration profile of AbNadE2. A) Elution profile of AbNadE2 on a Superose 6 column, the elution volume corresponds to 103 kDa. B) SDS-PAGE analysis of gel filtration chromatography fractions.

5. Fig. S5 Fig. S5– Superposition of the NAD+ synthetase domain of NadE2Gln from Burkholderiathailandensis(red) and NadENH3 from Bacillus subtilis(green). View from the top of the NAD+ synthetase domain. The PDB structures 4FH4 (amino acids 281 to 542) and 1KQP (full length) were aligned and visualized using Pymol.