<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

Folding Mechanism of an Extremely Thermostable (βα)₈-Barrel Enzyme: A High Kinetic Barrier Protects the Protein from Denaturation

HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from <i>Thermotoga maritima</i>, is an extremely thermostable (βα)₈-barrel protein. We elucidated the unfolding and refolding mechanism of HisF. Its unfolding transition is reversible and adequately described by the two-state model, but 6 weeks is necessary to reach equilibrium (at 25 °C). During refolding, initially a burst-phase off-pathway intermediate is formed. The subsequent productive folding occurs in two kinetic phases with time constants of ∼3 and ∼20 s. They reflect a sequential process via an on-pathway intermediate, as revealed by stopped-flow double-mixing experiments. The final step leads to native HisF, which associates with the glutaminase subunit HisH to form the functional ImGPS complex. The conversion of the on-pathway intermediate to the native protein results in a 10⁶-fold increase of the time constant for unfolding from 89 ms to 35 h (at 4.0 M GdmCl) and thus establishes a high energy barrier to denaturation. We conclude that the extra stability of HisF is used for kinetic protection against unfolding. In its refolding mechanism, HisF resembles other (βα)₈-barrel proteins

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

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

Constitutively Active Glutaminase Variants Provide Insights into the Activation Mechanism of Anthranilate Synthase

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys–His–Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys–His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism

Performance of SSD and MSD on the NMR ensemble hIFABP.

<p>enzdes (blue lines) was executed for 1000 runs <i>i</i> for each of the ten conformations in the ensemble. For each number of runs <i>i</i>, the value (dotted line) is the mean of the ten lowest-energy sequences (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005600#pcbi.1005600.e029" target="_blank">Eq 6</a>). The corresponding value (solid line) is the mean recovery value deduced from the same sequences (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005600#pcbi.1005600.e026" target="_blank">Eq 5</a>). MSF:GA:enzdes (orange lines) was carried out for 800 generations <i>j</i> on the whole ensemble using a population of 210 sequences. For each generation <i>j</i>, the value (dotted line) is the mean of the ten lowest-energy sequences of the corresponding population (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005600#pcbi.1005600.e032" target="_blank">Eq 7</a>). The corresponding value (solid line) is the mean recovery value deduced from the same sequences (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005600#pcbi.1005600.e026" target="_blank">Eq 5</a>).</p

Mutations introduced into the IGPS scaffold to design retro-aldolase activity.

<p>(<b>a</b>) An overview of all mutations introduced in 42 previous designs subsumed in the set RA* which are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005600#pcbi.1005600.s003" target="_blank">S3 Text</a>. Blue spheres indicate residue positions and sphere diameters are proportional to the frequency of the mutations in comparison to the native IGPS sequence. (<b>b</b>) Ditto, for nine RA_MSD* designs, mutations are visualized by means of orange spheres.</p

Recovery of two striking binding pockets by means of enzdes and MSF:GA:enzdes.

<p><b>(a)</b> The 3D structure of the binding pocket of ARL3-GDP is shown on the right, the ligand GDP is colored light blue. The residues of the corresponding design positions are shown on the left (labeled “Native”). The sequence logos labeled enzdes and MSF:GA:enzdes represent for each design position the distribution of residues as generated by the corresponding protocols. Residues that are similar to the native ones are colored in green. In the native sequence, residues are colored in teal, if the outcome of the two protocols differs drastically. <b>(b)</b> The 3D structure of the binding pocket of the glucose binding protein is shown on the right; the bound glucose is colored light blue. Native residues and sequence logos are shown on the left and were prepared and colored as described for panel <b>(a)</b>.</p

Single-state designability of <i>MD_EnzBench</i> conformations.

<p>Each of the 100 boxplots on the right represents 16 × 10 <i>nssr</i> values resulting from ten conformations generated by the MD simulation in a 100 ps interval for each of the 16 <i>prot</i>(<i>k</i>). As a control, the 16 × 20 values of (single) enzdes designs generated for 20 protein-specific conformations from <i>BR_EnzBench</i> were summarized in a boxplot shown on the left (label Backrub). Whiskers indicate the lowest and the highest values of the 1.5 interquartile.</p