Fig. 3. Defining features of A. baumannii SpoT catalysis.

a, Surface representation of SpoT_Ab in the τ-state. The active site cavity in the HD domain is boxed in dashed lines. b, Zoom into the HD-active site of the SpoT_Ab–ppGpp complex. The acidic half of the interface (residues R45, Y51, E82, D83 and K140) and the Mn²⁺ ion activate the water molecule for nucleophilic attack of the pyrophosphate bond pf ppGpp, while the basic half of the interface (K46, K158 and R161) stabilizes the 3′ and 5′ phosphates of the alarmone substrate. c, Ribbon representation of the active site of SpoT_Ab revealing the residues involved in coordination of ppGpp. The unbiased mFo-DFc electron density map corresponding to these residues is shown in dark blue. d, Effects of Ala substitutions in the ppGpp binding site on the HD activity of SpoT_Ab. The residues for substitution were selected as per c. e, The HD functionality test of truncated versions of SpoT_Ab. SpoT_Ab variants (each labeled on the figure) were coexpressed expressed with RelA_Ab in ∆relA∆spoT Ptac::relA A. baumannii (AB5075). The ability of SpoT_Ab to promote the growth is reflective of its HD competence. f, HD activity of SpoT_Ab and the C-terminally truncated SpoT_Ab variants. Turnovers corresponding to each protein variant are colored as per the domain color code in e. Error bars represent s.d. of three or more independent samples examined over three independent experiments.