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A general and efficient strategy for generating the stable enzymes

View Article: PubMed Central - PubMed

ABSTRACT

The local flexibility of an enzyme’s active center plays pivotal roles in catalysis, however, little is known about how the flexibility of these flexible residues affects stability. In this study, we proposed an active center stabilization (ACS) strategy to improve the kinetic thermostability of Candida rugosa lipase1. Based on the B-factor ranking at the region ~10 Å within the catalytic Ser209, 18 residues were selected for site-saturation mutagenesis. Based on three-tier high-throughput screening and ordered recombination mutagenesis, the mutant VarB3 (F344I/F434Y/F133Y/F121Y) was shown to be the most stable, with a 40-fold longer in half-life at 60 °C and a 12.7 °C higher Tm value than that of the wild type, without a decrease in catalytic activity. Further analysis of enzymes with different structural complexities revealed that focusing mutations on the flexible residues within around 10 Å of the catalytic residue might increase the success rate for enzyme stabilization. In summary, this study identifies a panel of flexible residues within the active center that affect enzyme stability. This finding not only provides clues regarding the molecular evolution of enzyme stability but also indicates that ACS is a general and efficient strategy for exploring the functional robustness of enzymes for industrial applications.

No MeSH data available.


(A) Prediction of normalized B-factors of WT (black) and the VarB3 mutant (red) and (B) Stereoview of the superimposed structures of WT (gray) and the VarB3 mutant (green). Residues Y121, Y133, I344 and Y434 are labeled in yellow, blue, green and marine, respectively. The catalytic residue Ser209 is labeled in pink. Symbols: red, oxygen atom; blue, nitrogen atom.
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f6: (A) Prediction of normalized B-factors of WT (black) and the VarB3 mutant (red) and (B) Stereoview of the superimposed structures of WT (gray) and the VarB3 mutant (green). Residues Y121, Y133, I344 and Y434 are labeled in yellow, blue, green and marine, respectively. The catalytic residue Ser209 is labeled in pink. Symbols: red, oxygen atom; blue, nitrogen atom.

Mentions: To investigate the rigidity of the active center, the PredyFlexy web server was used to predict the normalized B-factor of the best mutant, VarB332. The normalized B-factors of all of the selected flexible residues are listed in Supplementary Table S3. As shown in Fig. 6A, the VarB3 mutant led to an increase in the rigidity of the region from 122 to 136, the region from 343 to 345, and the region from 432 to 436. All of these regions are near the mutated sites of residues 121, 133, 344 and 434.


A general and efficient strategy for generating the stable enzymes
(A) Prediction of normalized B-factors of WT (black) and the VarB3 mutant (red) and (B) Stereoview of the superimposed structures of WT (gray) and the VarB3 mutant (green). Residues Y121, Y133, I344 and Y434 are labeled in yellow, blue, green and marine, respectively. The catalytic residue Ser209 is labeled in pink. Symbols: red, oxygen atom; blue, nitrogen atom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036031&req=5

f6: (A) Prediction of normalized B-factors of WT (black) and the VarB3 mutant (red) and (B) Stereoview of the superimposed structures of WT (gray) and the VarB3 mutant (green). Residues Y121, Y133, I344 and Y434 are labeled in yellow, blue, green and marine, respectively. The catalytic residue Ser209 is labeled in pink. Symbols: red, oxygen atom; blue, nitrogen atom.
Mentions: To investigate the rigidity of the active center, the PredyFlexy web server was used to predict the normalized B-factor of the best mutant, VarB332. The normalized B-factors of all of the selected flexible residues are listed in Supplementary Table S3. As shown in Fig. 6A, the VarB3 mutant led to an increase in the rigidity of the region from 122 to 136, the region from 343 to 345, and the region from 432 to 436. All of these regions are near the mutated sites of residues 121, 133, 344 and 434.

View Article: PubMed Central - PubMed

ABSTRACT

The local flexibility of an enzyme’s active center plays pivotal roles in catalysis, however, little is known about how the flexibility of these flexible residues affects stability. In this study, we proposed an active center stabilization (ACS) strategy to improve the kinetic thermostability of Candida rugosa lipase1. Based on the B-factor ranking at the region ~10 Å within the catalytic Ser209, 18 residues were selected for site-saturation mutagenesis. Based on three-tier high-throughput screening and ordered recombination mutagenesis, the mutant VarB3 (F344I/F434Y/F133Y/F121Y) was shown to be the most stable, with a 40-fold longer in half-life at 60 °C and a 12.7 °C higher Tm value than that of the wild type, without a decrease in catalytic activity. Further analysis of enzymes with different structural complexities revealed that focusing mutations on the flexible residues within around 10 Å of the catalytic residue might increase the success rate for enzyme stabilization. In summary, this study identifies a panel of flexible residues within the active center that affect enzyme stability. This finding not only provides clues regarding the molecular evolution of enzyme stability but also indicates that ACS is a general and efficient strategy for exploring the functional robustness of enzymes for industrial applications.

No MeSH data available.