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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.


Related in: MedlinePlus

The topological structures of monomeric globular lipases LipA (A), CalB (B), and LIP1 (C). α-helices and β-strands are shown as cylinders and arrows, respectively. The canonical α/β-hydrolase folds are in red. The lids of LIP1 and CalB are shown in yellow. Other structures in the catalytic domains are shown in white. All serine catalytic residues are represented as green sticks. The blue rings represent the range of 10 Å around the catalytic residue serine.
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f1: The topological structures of monomeric globular lipases LipA (A), CalB (B), and LIP1 (C). α-helices and β-strands are shown as cylinders and arrows, respectively. The canonical α/β-hydrolase folds are in red. The lids of LIP1 and CalB are shown in yellow. Other structures in the catalytic domains are shown in white. All serine catalytic residues are represented as green sticks. The blue rings represent the range of 10 Å around the catalytic residue serine.

Mentions: The B-factor (or B-value), indicating atomic displacement parameters obtained from X-ray data, is commonly used to represent residue flexibility11. Flexible residues, which have fewer contacts with other amino acids, may produce local perturbations inside highly complex networks of non-covalent connections. The residues trigger protein unfolding due to their large thermal fluctuations. Hence, introducing mutations to rigidify flexible residues may be an effective way to improve enzyme thermostability12. Based on the B-factor, Reetz et al.13 significantly increased the thermostability of lipase ( value of 45 °C) from Bacillus subtilis (LipA, 181 residues) (Fig. 1A), which is a minimal α/β hydrolase14. Later, Zhang et al.15 also reported a double-mutant of Rhizomucor miehei lipase (RML, 269 residues) based on B-factor design but only achieved a 1.24-fold thermostability increase. Damnjanović et al.16 mutated 12 residues with the highest B-factors on the surface of phosphatidylinositol-synthesizing Streptomyces phospholipase D (PLD, 509 residues), and the half-life of the best mutant was only 8.7 min longer compared to the parent (25.9 min). All of these results suggest that rigidifying flexible residues on larger enzymes based on only B-factor analysis, without considering other structural factors, may have a limitation in enhancing the stability of a variety of enzymes.


A general and efficient strategy for generating the stable enzymes
The topological structures of monomeric globular lipases LipA (A), CalB (B), and LIP1 (C). α-helices and β-strands are shown as cylinders and arrows, respectively. The canonical α/β-hydrolase folds are in red. The lids of LIP1 and CalB are shown in yellow. Other structures in the catalytic domains are shown in white. All serine catalytic residues are represented as green sticks. The blue rings represent the range of 10 Å around the catalytic residue serine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: The topological structures of monomeric globular lipases LipA (A), CalB (B), and LIP1 (C). α-helices and β-strands are shown as cylinders and arrows, respectively. The canonical α/β-hydrolase folds are in red. The lids of LIP1 and CalB are shown in yellow. Other structures in the catalytic domains are shown in white. All serine catalytic residues are represented as green sticks. The blue rings represent the range of 10 Å around the catalytic residue serine.
Mentions: The B-factor (or B-value), indicating atomic displacement parameters obtained from X-ray data, is commonly used to represent residue flexibility11. Flexible residues, which have fewer contacts with other amino acids, may produce local perturbations inside highly complex networks of non-covalent connections. The residues trigger protein unfolding due to their large thermal fluctuations. Hence, introducing mutations to rigidify flexible residues may be an effective way to improve enzyme thermostability12. Based on the B-factor, Reetz et al.13 significantly increased the thermostability of lipase ( value of 45 °C) from Bacillus subtilis (LipA, 181 residues) (Fig. 1A), which is a minimal α/β hydrolase14. Later, Zhang et al.15 also reported a double-mutant of Rhizomucor miehei lipase (RML, 269 residues) based on B-factor design but only achieved a 1.24-fold thermostability increase. Damnjanović et al.16 mutated 12 residues with the highest B-factors on the surface of phosphatidylinositol-synthesizing Streptomyces phospholipase D (PLD, 509 residues), and the half-life of the best mutant was only 8.7 min longer compared to the parent (25.9 min). All of these results suggest that rigidifying flexible residues on larger enzymes based on only B-factor analysis, without considering other structural factors, may have a limitation in enhancing the stability of a variety of 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.


Related in: MedlinePlus