Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed.


Abstract

Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.

Submission Details

ID: AJSM4jY7

Submitter: Shu-Ching Ou

Submission Date: Oct. 10, 2018, 12:31 p.m.

Version: 1

Publication Details
Carlin DA;Hapig-Ward S;Chan BW;Damrau N;Riley M;Caster RW;Bethards B;Siegel JB,PLoS One (2017) Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed. PMID:28531185
Additional Information

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
2O9P 2007-10-02 2.1 beta-glucosidase B from Paenibacillus polymyxa
2O9T 2007-10-02 2.15 beta-glucosidase B from Bacillus polymyxa complexed with glucose
2O9R 2007-10-02 2.3 beta-glucosidase B complexed with thiocellobiose
2JIE 2007-07-03 2.3 BETA-GLUCOSIDASE B FROM BACILLUS POLYMYXA COMPLEXED WITH 2-F-GLUCOSE
2Z1S 2007-10-02 2.46 Beta-glucosidase B from paenibacillus polymyxa complexed with cellotetraose

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Beta-glucosidase B P22505 BGLB_PAEPO