<i>In vivo</i> complementation of auxotrophic <i>E</i>. <i>coli</i> strains by PriA, HisA, HisA ancestors, and TrpF.

<p><i>In vivo</i> complementation of auxotrophic <i>E</i>. <i>coli</i> strains by PriA, HisA, HisA ancestors, and TrpF.</p

Steady-state kinetic parameters of extant PriA and HisA enzymes, and reconstructed HisA ancestors.

<p>Steady-state kinetic parameters of extant PriA and HisA enzymes, and reconstructed HisA ancestors.</p

Phylogenetic tree depicting the position of extant HisA and PriA enzymes (diamonds) and their relationship to the reconstructed ancestral HisA enzymes (circles).

<p>The topology of the tree was inferred from the phylogenetic trees used for sequence reconstruction (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005836#pgen.1005836.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005836#pgen.1005836.s002" target="_blank">S2</a> Figs). CA-Act-HisA, CA-Prot-HisA, and CA-Bact-HisA are the predecessor of HisA enzymes from Actinobacteria, Proteobacteria and Bacteria, respectively. Note that actinobacterial sequences were omitted for reconstruction of CA-Prot-HisA and CA-Bact-HisA (indicated by grey shading of the Actinobacteria branch). ddHisA and tmHisA were not used for sequence reconstruction and are only listed because they were characterized experimentally. The vertical bar indicates the branch length that corresponds to 0.5 mutations per site. The catalytic efficiencies <i>k</i>_cat/<i>K</i>_M of the enzymes for processing ProFAR and PRA are given in red and blue, respectively. Abbreviations: sc, <i>S</i>. <i>coelicolor</i>; dd, <i>D</i>. <i>desulfuricans</i>; pp, <i>P</i>. <i>carbinolicus</i>; tm, <i>T</i>. <i>maritima</i>; Sp., Spirochaetes; Bact., Bacteroidetes.</p

Two states of the PriA active site from <i>M</i>. <i>tuberculosis</i>.

<p>(a) Schematic view of the site in the HisA-state (bound product PRFAR, PDB ID 3zs4). (b) The same active site in the TrpF-state (bound product analogue reduced-CdRP, PDB ID 2y85). Residues of the active site are shown as stick models. Residue numbering is based on PDB ID 3zs4. (c) Sequence logos showing the conservation of the motif as deduced from SSN clusters of the HisA/PriA superfamily. Basic and acidic residues are colored blue and red, respectively.</p

Evidence for the Existence of Elaborate Enzyme Complexes in the Paleoarchean Era

Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (βα)₈-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (βα)₈-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago