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Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

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Activity of ligase enzymes assayed at different temperatures.Ligases #6 and #7 had been selected previously at 23°C [17], [22] and ligase 10C was selected at 65°C. In this assay, the 32P-labeled PPP-substrate-65, HO-substrate-65 and 40 nt splint were incubated with the individual enzymes for 16 h and the activity was monitored by a gel-shift assay.
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pone-0112028-g004: Activity of ligase enzymes assayed at different temperatures.Ligases #6 and #7 had been selected previously at 23°C [17], [22] and ligase 10C was selected at 65°C. In this assay, the 32P-labeled PPP-substrate-65, HO-substrate-65 and 40 nt splint were incubated with the individual enzymes for 16 h and the activity was monitored by a gel-shift assay.

Mentions: To compare the enzymatic activity of ligase 10C to ligases #6 and #7, we assayed the three enzymes at 23°C and 65°C (Figure 4). Ligase 10C was the only enzyme active at 65°C. In comparison, ligases #6 and #7 were active at room temperature as expected, but had no measurable activity at 65°C. In addition to its activity at 65°C, ligase 10C was also active at room temperature. To compare the activity of the three enzymes more accurately, we measured the kobs for each ligase at 23°C. At a subsaturating substrate concentration of 10 µM, ligase 10C had a kobs of 0.165±0.015 h−1 while ligases #6 and #7 had kobs of 0.0174±0.0066 h−1 and kobs of 0.0207±0.0045 h−1, respectively (Table S1). This represents an 8 to 10-fold increased activity of ligase 10C compared to ligases #6 and #7 even at 23°C. While the main goal of the selection was to isolate an enzyme with greater thermostability, as an added benefit, the most stable enzyme also featured an improved catalytic rate at room temperature.


Thermostable artificial enzyme isolated by in vitro selection.

Morelli A, Haugner J, Seelig B - PLoS ONE (2014)

Activity of ligase enzymes assayed at different temperatures.Ligases #6 and #7 had been selected previously at 23°C [17], [22] and ligase 10C was selected at 65°C. In this assay, the 32P-labeled PPP-substrate-65, HO-substrate-65 and 40 nt splint were incubated with the individual enzymes for 16 h and the activity was monitored by a gel-shift assay.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230948&req=5

pone-0112028-g004: Activity of ligase enzymes assayed at different temperatures.Ligases #6 and #7 had been selected previously at 23°C [17], [22] and ligase 10C was selected at 65°C. In this assay, the 32P-labeled PPP-substrate-65, HO-substrate-65 and 40 nt splint were incubated with the individual enzymes for 16 h and the activity was monitored by a gel-shift assay.
Mentions: To compare the enzymatic activity of ligase 10C to ligases #6 and #7, we assayed the three enzymes at 23°C and 65°C (Figure 4). Ligase 10C was the only enzyme active at 65°C. In comparison, ligases #6 and #7 were active at room temperature as expected, but had no measurable activity at 65°C. In addition to its activity at 65°C, ligase 10C was also active at room temperature. To compare the activity of the three enzymes more accurately, we measured the kobs for each ligase at 23°C. At a subsaturating substrate concentration of 10 µM, ligase 10C had a kobs of 0.165±0.015 h−1 while ligases #6 and #7 had kobs of 0.0174±0.0066 h−1 and kobs of 0.0207±0.0045 h−1, respectively (Table S1). This represents an 8 to 10-fold increased activity of ligase 10C compared to ligases #6 and #7 even at 23°C. While the main goal of the selection was to isolate an enzyme with greater thermostability, as an added benefit, the most stable enzyme also featured an improved catalytic rate at room temperature.

Bottom Line: This process commonly results in a simultaneous reduction of protein stability as an undesired side effect.Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes.These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America, & BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America.

ABSTRACT
Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10 C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10 C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10 C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.

Show MeSH
Related in: MedlinePlus