(A) A maximum likelihood phylogenetic tree of 430 HPRT amino acid sequences rooted on eukaryotic HPRTs. Bootstrap values from 100 replicates are shown. Anc1-3 refer to ancestral HPRTs. The tree reveals two main lineages: one with dimer-dimer interaction motifs (Anc2; purple) and one lacking the interaction motifs (Anc3; blue). Branch length scale indicates 0.5 substitutions per site. See Materials and methods for tree construction. See Source Data one for the full alignment of HPRTs. (B) Alignment of HPRT dimer-dimer interface from Anc1, Anc2, Anc3, and select extant HPRTs. Nearly all (p)ppGpp-regulated HPRTs share Lys81 found in Anc2. Dimeric HPRTs insensitive to basal (p)ppGpp lack Lys81, but share Trp82, Pro86, and Ala110 (B. anthracis Hpt-1 numbering). Figure 6—figure supplement 1 shows the role of these residues at the interface. For the ancestors, red indicates high (>90%), yellow moderate (50–90%), and no color low (<50%) confidence level in the residue identity. (C) B. subtilis K81E and K81A HPRTs have weakened tetramerization according to size-exclusion chromatography. Figure 6—figure supplement 2 shows Lys81 at the dimer–dimer interface. (D) Binding of 32P-labeled pppGpp to wild type (black circles), K81A (red squares), and K81E (blue triangles) HPRTs using DRaCALA. Error bars represent SEM of three replicates. (E) PRPP competition with 32P-labeled pppGpp for binding wild type (black circles) and K81A HPRTs (red squares). K81A is more sensitive to PRPP competition than the tetrameric wild type. Error bars are range of duplicate.