Figure 2 |. Tas1_tox adopts a RelA-SpoT Homolog (RSH) fold found in enzymes that synthesize the bacterial alarmone (p)ppGpp but instead synthesizes (p)ppApp.

a) Overall structure of PA14_01140_tox in complex with PA14_01130. Shown are ribbon (left) and space-filling (right) representations of PA14_01140_tox (purple) in complex with PA14_01130 (green). b) PA14_01140_tox resembles (p)ppGpp synthetase enzymes. Structural overlay of PA14_01140_tox and the small alarmone synthetase RelQ from Bacillus subtilis (PDB code 5DEC)²². The structures superimpose with a Cα r.s.m.d. of 3.4Å over 145 equivalent positions. c) Structural alignment of the pyrophosphate donor ATP binding site of RelQ in complex with a magnesium ion and the non-hydrolyzable ATP analog AMPCPP (PDB code 5F2V) with the equivalent amino acid positions in PA14_01140_tox. Amino acid side chains deriving from PA14_01140_tox or RelQ and their corresponding labels are shown in purple and yellow, respectively. d) PA14_01140_tox catalyzes the formation of AMP in a dose-dependent manner. Coupled enzyme assay of PA14_01140_tox-catalyzed AMP production as a function of NADH consumption over time. e) PA14_01140_tox catalyzes the production of AMP from ATP in a GTP-independent manner. The control reaction lacks adenylate kinase, which is required for the initial step of the coupled assay. f) PA14_01140_tox (Tas1_tox) is a (p)ppApp synthetase enzyme. Anion-exchange traces of ATP alone or with excess AMP or ADP after incubation with Tas1_tox. A standard trace for ATP, ADP and AMP is shown for comparison. g) Rate of ppApp production by Tas1_tox or Tas1_tox^E382A. Reactions were performed at 37°C with 10 mM ATP and 1 nM Tas1_tox or 1 μM Tas1_tox^E382A. Data are mean ±SD from three separate reactions. h) Specificity of Tas1_tox towards pyrophosphate (PP_i) donors and acceptors. Indicated nucleotides (1 mM each) were incubated with 100 nM Tas1_tox at room temperature for 10 minutes. Reactions that progressed to completion (++), made detectable product (+) or made no detectable product (−) are indicated. NT, not tested. See also Extended Data Figure 6. d–f) Data are representative of two technical replicates.