Enhanced enzyme kinetic stability by increasing rigidity within the active site.


Abstract

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

Submission Details

ID: KKnySLt73

Submitter: Shu-Ching Ou

Submission Date: Jan. 30, 2019, 5:25 p.m.

Version: 1

Publication Details
Xie Y;An J;Yang G;Wu G;Zhang Y;Cui L;Feng Y,J Biol Chem (2014) Enhanced enzyme kinetic stability by increasing rigidity within the active site. PMID:24448805
Additional Information

Due to numbering (starts from -1 in PDB) issue, D223G/L278M/A281E are shown as D225G/L280M/A283E. Variant A281E(A283E) was deactivated too rapidly to measure at 48 °C. The half-life was less than 5 min at only 45 °C.

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5A71 2015-10-21 0.91 Open and closed conformations and protonation states of Candida antarctica Lipase B: atomic resolution native
4K5Q 2014-01-29 1.49 Crystal structure of CALB mutant DGLM from Candida antarctica
3ICV 2009-10-06 1.49 Structural Consequences of a Circular Permutation on Lipase B from Candida Antartica
4K6G 2014-01-29 1.5 Crystal structure of CALB from Candida antarctica
1TCA 1994-05-31 1.55 THE SEQUENCE, CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT OF TWO CRYSTAL FORMS OF LIPASE B FROM CANDIDA ANTARCTICA
4K6H 2014-01-29 1.6 Crystal structure of CALB mutant L278M from Candida antarctica
4K6K 2014-01-29 1.6 Crystal structure of CALB mutant D223G from Candida antarctica
3ICW 2009-10-06 1.69 Structure of a Circular Permutation on Lipase B from Candida Antartica with Bound Suicide Inhibitor
6ISQ 2019-07-24 1.86 structure of Lipase mutant with oxided Cys-His-Asp catalytic triad
6ISP 2019-07-24 1.88 structure of Candida antarctica Lipase B mutant
4ZV7 2016-01-13 2.0 Crystal structure of hexagonal form of lipase B from Candida antarctica
1TCB 1994-05-31 2.1 THE SEQUENCE, CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT OF TWO CRYSTAL FORMS OF LIPASE B FROM CANDIDA ANTARCTICA
5A6V 2015-10-21 2.28 Open and closed conformations and protonation states of Candida antarctica Lipase B: Xenon complex
1TCC 1994-05-31 2.5 THE SEQUENCE, CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT OF TWO CRYSTAL FORMS OF LIPASE B FROM CANDIDA ANTARCTICA
1LBT 1995-10-15 2.5 LIPASE (E.C.3.1.1.3) (TRIACYLGLYCEROL HYDROLASE)
1LBS 1995-10-15 2.6 LIPASE (E.C.3.1.1.3) (TRIACYLGLYCEROL HYDROLASE)
6ISR 2019-07-24 2.6 structure of lipase mutant with Cys-His-Asp catalytic triad
3W9B 2014-04-02 2.9 Crystal structure of Candida antarctica lipase B with anion-tag

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
200.0 A,B Lipase B P41365 LIPB_PSEA2