Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins.

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.

Assessment and Impacts of Phosphorylation on Protein Flexibility of the α-d-Phosphohexomutases.

Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in the enzyme mechanism, the phosphorylation state of the catalytic phosphoserine has recently been found to have widespread effects on the structural flexibility of enzymes in this superfamily. These effects must be carefully accounted for when assessing other perturbations to these enzymes, such as mutations or ligand binding. In this chapter, we focus on methods for assessing and modulating the phosphorylation state of the catalytic serine, as well as straightforward ways to probe the impacts of this modification on protein structure/flexibility. This knowledge is essential for producing homogeneous and stable samples of these proteins for biophysical studies. The methods described herein should be widely applicable to enzymes across the PHM superfamily and may also be useful in characterizing the effects of posttranslational modifications on other proteins.

Dynamics of the excised base release in thymine DNA glycosylase during DNA repair process.

Thymine DNA glycosylase (TDG) initiates base excision repair by cleaving the N-glycosidic bond between the sugar and target base. After catalysis, the release of excised base is a requisite step to terminate the catalytic cycle and liberate the TDG for the following enzymatic reactions. However, an atomistic-level understanding of the dynamics of the product release process in TDG remains unknown. Here, by employing molecular dynamics simulations combined with the Markov State Model, we reveal the dynamics of the thymine release after the excision at microseconds timescale and all-atom resolution. We identify several key metastable states of the thymine and its dominant releasing pathway. Notably, after replacing the TDG residue Gly142 with tyrosine, the thymine release is delayed compared to the wild-type (wt) TDG, as supported by our potential of mean force (PMF) calculations. These findings warrant further experimental tests to potentially trap the excised base in the active site of TDG after the catalysis, which had been unsuccessful by previous attempts. Finally, we extended our studies to other TDG products, including the uracil, 5hmU, 5fC and 5caC bases in order to compare the product release for different targeting bases in the TDG-DNA complex.

T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA-DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.

Bacterial Expression and HTS Assessment of Soluble Epoxide Hydrolase Phosphatase.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were identified.

Characterization of catalytic carboxylate triad in non-replicative DNA polymerase III (pol E) of Geobacillus kaustophilus HTA.

Three aspartic acid residues D378, D380 and D531 form the catalytic carboxylate triad in Geobacillus kaustophilus (Gka) DNA polymerase III α-subunit homolog, pol E. We cloned, expressed and purified wild type (WT), alanine (D → A) and glutamate (D → E) mutant enzymes of D378, D380 and D531. The WT and mutant enzymes were biochemically characterized for DNA binding, dNTP binding and catalytic activity in the presence of two metal ions (Mg2+ and Mn2+). The polymerase activity of all mutant enzymes was lost in the presence Mg2+, whereas D378E and D531E mutant enzymes showed about 35 and 60 percent activity, with Mn2+. D380E mutant enzyme did not show noticeable activity with either metal ions suggesting its absolute requirement in polymerase reaction. Kinetic characterization of individual mutant proteins showed that the template-primer binding affinity (KD.DNA) did not change due to both D → A or D → E mutation. The KM.dNTP for D378E and D531E increased by about 10- and 100-fold, compared to WT enzyme implicating the function of these residues in dNTP binding. Based on these results and the analysis of the available crystal structures of the homologous enzyme species in their apo and E.DNA.dNTP ternary complex forms, we conclude that D378 and D531 are mainly responsible for the binding of metal chelated substrate dNTP, while D380 is solely responsible for the chemical step of phosphodiester bond formation.

Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies.

Glycoside hydrolase family 13 (GH13) members have evolved to possess various distinct reaction specificities despite the overall structural similarity. In this study we investigated the evolutionary input required to effeciently interchange these specificities and also compared the effectiveness of laboratory evolution techniques applied, i.e., error-prone PCR and saturation mutagenesis. Conversion of our model enzyme, cyclodextrin glucanotransferase (CGTase), into an alpha-amylase like hydrolytic enzyme by saturation mutagenesis close to the catalytic core yielded a triple mutant (A231V/F260W/F184Q) with the highest hydrolytic rate ever recorded for a CGTase, similar to that of a highly active alpha-amylase, while cyclodextrin production was virtually abolished. Screening of a much larger, error-prone PCR generated library yielded far less effective mutants. Our results demonstrate that it requires only three mutations to change CGTase reaction specificity into that of another GH13 enzyme. This suggests that GH13 members may have diversified by introduction of a limited number of mutations to the common ancestor, and that interconversion of reaction specificites may prove easier than previously thought.