Effects of Random Mutagenesis and In Vivo Selection on the Specificity and Stability of a Thermozyme


Abstract

Factors that give enzymes stability, activity, and substrate recognition result from the combination of few weak molecular interactions, which can be difficult to study through rational protein engineering approaches. We used irrational random mutagenesis and in vivo selection to test if a β-glycosidase from the thermoacidophile Saccharolobus solfataricus (Ssβ-gly) could complement an Escherichia coli strain unable to grow on lactose. The triple mutant of Ssβ-gly (S26L, P171L, and A235V) was more active than the wild type at 85 °C, inactivated at this temperature almost 300-fold quicker, and showed a 2-fold higher kcat on galactosides. The three mutations, which were far from the active site, were analyzed to test their effect at the structural level. Improved activity on galactosides was induced by the mutations. The S26L and P171L mutations destabilized the enzyme through the removal of a hydrogen bond and increased flexibility of the peptide backbone, respectively. However, the flexibility added by S26L mutation improved the activity at T > 60 °C. This study shows that random mutagenesis and biological selection allowed the identification of residues that are critical in determining thermal activity, stability, and substrate recognition.

Submission Details

ID: TPwBQTuE4

Submitter: Shu-Ching Ou

Submission Date: June 24, 2019, 11:30 a.m.

Version: 1

Publication Details
Perugino G;Strazzulli A;Mazzone M;Rossi M;Moracci M,Catalysts (2019) Effects of Random Mutagenesis and In Vivo Selection on the Specificity and Stability of a Thermozyme
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
1GOW 1996-09-19T00:00:00+0000 2.6 BETA-GLYCOSIDASE FROM SULFOLOBUS SOLFATARICUS
2CEQ 2006-02-10T00:00:00+0000 2.14 Beta-glycosidase from Sulfolobus solfataricus in complex with glucoimidazole
2CER 2006-02-10T00:00:00+0000 2.29 Beta-glycosidase from Sulfolobus solfataricus in complex with phenethyl-substituted glucoimidazole
1UWQ 2004-02-11T00:00:00+0000 2.02 Structure of beta-glycosidase from Sulfolobus solfataricus
1UWU 2004-02-11T00:00:00+0000 1.95 Structure of beta-glycosidase from Sulfolobus solfataricus in complex with D-glucohydroximo-1,5-lactam
1UWR 2004-02-11T00:00:00+0000 2.14 Structure of beta-glycosidase from Sulfolobus solfataricus in complex with 2-deoxy-2-fluoro-galactose
1UWI 2004-02-05T00:00:00+0000 2.55 CRYSTAL STRUCTURE OF MUTATED BETA-GLYCOSIDASE FROM SULFOLOBUS SOLFATARICUS, WORKING AT MODERATE TEMPERATURE
1UWS 2004-02-11T00:00:00+0000 1.95 Structure of beta-glycosidase from Sulfolobus solfataricus in complex with 2-deoxy-2-fluoro-glucose
1UWT 2004-02-11T00:00:00+0000 1.95 Structure of beta-glycosidase from Sulfolobus solfataricus in complex with D-galactohydroximo-1,5-lactam
5IXE 2016-03-23T00:00:00+0000 1.75 1.75A RESOLUTION STRUCTURE OF 5-Fluoroindole BOUND BETA-GLYCOSIDASE (W33G) FROM SULFOLOBUS SOLFATARICUS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.7 Beta-galactosidase P50388 BGAL_SACSH
100.0 Beta-galactosidase P22498 BGAL_SACS2