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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.


Model structures of WT and the VarB3 mutant (F344I/F434Y/F133Y/F121Y) based on PDB: 1CRL.(A) Comparison of the structural changes in the loop region from 120 to 136 which included the mutant position 121 and 133. (B) Comparison of α12 structural changes around mutant position 344. (C) Intramolecular interactions near mutant position 434 in WT and VarB3 mutant. The new generated hydrogen bonds are indicated by purple lines and other hydrogen bonds are represented by yellow dashed lines. Symbols: red, oxygen atom; blue, nitrogen atom.
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f7: Model structures of WT and the VarB3 mutant (F344I/F434Y/F133Y/F121Y) based on PDB: 1CRL.(A) Comparison of the structural changes in the loop region from 120 to 136 which included the mutant position 121 and 133. (B) Comparison of α12 structural changes around mutant position 344. (C) Intramolecular interactions near mutant position 434 in WT and VarB3 mutant. The new generated hydrogen bonds are indicated by purple lines and other hydrogen bonds are represented by yellow dashed lines. Symbols: red, oxygen atom; blue, nitrogen atom.

Mentions: The modeling structure of the VarB3 mutant was constructed using Discover Studio3.5 and was assessed using PROCHECK analysis33, which revealed that only one residue (Ile18) was in a disallowed region in the Ramachandran Plot, similar to the wild type (Supplementary Fig. S3). We observed no gross changes in the structure of the VarB3 mutant compared to WT (Fig. 6B). The mutated positions at 121 and 133 were located in the same loop region from 120 to 136, and positions 344 and 434 were located in α12 and β13, respectively. However, the model structure showed that the Tyr133 residue formed 3 hydrogen bonds with two water molecules (Wa and Wb) and Gly122, generating a new hydrogen-bond network (Fig. 7A). The Tyr121 residue formed 4 hydrogen bonds with water molecule Wc, Phe128, Asn155 and Tyr156 (Fig. 7A). The new hydrogen-bond networks may enhance the stability of the loop region from 120 to 136. The Ile344 residue shortened the distance between Thr343 and Phe345, which resulted in the formation of a 3.3 Å hydrogen bond between them (Fig. 7B). Furthermore, the lengths of the 3 hydrogen bonds between Phe345 and Ser348, Gly346 and Ser349 (2 hydrogen bonds), respectively, became shorter from 3.3 Å to 3.2 Å. Compared to the structure of WT, the structural flexibility around position 344 was significantly increased, which was annotated by a prolonged α12 helix. The Tyr434 residue led to that the generation of a new hydrogen bond with the water molecule Wd, and the distances of some hydrogen bonds around β13 were shortened, thus contributing to a stabilized LIP1 (Fig. 7C).


A general and efficient strategy for generating the stable enzymes
Model structures of WT and the VarB3 mutant (F344I/F434Y/F133Y/F121Y) based on PDB: 1CRL.(A) Comparison of the structural changes in the loop region from 120 to 136 which included the mutant position 121 and 133. (B) Comparison of α12 structural changes around mutant position 344. (C) Intramolecular interactions near mutant position 434 in WT and VarB3 mutant. The new generated hydrogen bonds are indicated by purple lines and other hydrogen bonds are represented by yellow dashed lines. 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

f7: Model structures of WT and the VarB3 mutant (F344I/F434Y/F133Y/F121Y) based on PDB: 1CRL.(A) Comparison of the structural changes in the loop region from 120 to 136 which included the mutant position 121 and 133. (B) Comparison of α12 structural changes around mutant position 344. (C) Intramolecular interactions near mutant position 434 in WT and VarB3 mutant. The new generated hydrogen bonds are indicated by purple lines and other hydrogen bonds are represented by yellow dashed lines. Symbols: red, oxygen atom; blue, nitrogen atom.
Mentions: The modeling structure of the VarB3 mutant was constructed using Discover Studio3.5 and was assessed using PROCHECK analysis33, which revealed that only one residue (Ile18) was in a disallowed region in the Ramachandran Plot, similar to the wild type (Supplementary Fig. S3). We observed no gross changes in the structure of the VarB3 mutant compared to WT (Fig. 6B). The mutated positions at 121 and 133 were located in the same loop region from 120 to 136, and positions 344 and 434 were located in α12 and β13, respectively. However, the model structure showed that the Tyr133 residue formed 3 hydrogen bonds with two water molecules (Wa and Wb) and Gly122, generating a new hydrogen-bond network (Fig. 7A). The Tyr121 residue formed 4 hydrogen bonds with water molecule Wc, Phe128, Asn155 and Tyr156 (Fig. 7A). The new hydrogen-bond networks may enhance the stability of the loop region from 120 to 136. The Ile344 residue shortened the distance between Thr343 and Phe345, which resulted in the formation of a 3.3 Å hydrogen bond between them (Fig. 7B). Furthermore, the lengths of the 3 hydrogen bonds between Phe345 and Ser348, Gly346 and Ser349 (2 hydrogen bonds), respectively, became shorter from 3.3 Å to 3.2 Å. Compared to the structure of WT, the structural flexibility around position 344 was significantly increased, which was annotated by a prolonged α12 helix. The Tyr434 residue led to that the generation of a new hydrogen bond with the water molecule Wd, and the distances of some hydrogen bonds around β13 were shortened, thus contributing to a stabilized LIP1 (Fig. 7C).

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.