Sortase mutants with improved protein thermostability and enzymatic activity obtained by consensus design

Abstract

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme’s stability while at the same time enhancing the enzyme’s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition

“…2 A ). The former is an example of a FRET quencher probe that is commonly used for monitoring sortase enzymatic activity ( 9 , 15 , 23 , 29 , 30 , 31 ), whereas the latter is a simplified target containing an acetyl ( Ac -) cap and C-terminal primary amide (- NH 2 ). For both substrates, LC–ESI–MS was used to confirm that they were cleaved by wildtype spySrtA in a model transacylation reaction ( Fig.…”

Engineered Sortases in Peptide and Protein Chemistry

“…[24] To improve the thermostability of Sa-SrtA, multiple sequence alignments of sortases revealed certain positions of sortases from extremophiles to be conserved or distinct from Sa-SrtA, identifying M155V and V193R as mutations that increased activity at 37°C or higher (Figure 2d; Table 1). [36] Notably, sortases evolved for high catalytic activity commonly come at the cost of decreased thermostability. It was therefore attempted to improve the stability of the activityenhanced M4 mutant from the original yeast display screen (Table 1).…”

“…To improve the thermostability of Sa‐SrtA, multiple sequence alignments of sortases revealed certain positions of sortases from extremophiles to be conserved or distinct from Sa‐SrtA, identifying M155V and V193R as mutations that increased activity at 37 °C or higher (Figure 2d; Table 1). [36] …”

Improved Synthesis of Furfurylamine from a High Titer of Biomass-Derived Furfural by a Thermostable Triple Mutant ω-Transaminase in a Three-Component Deep Eutectic Solvent ChCl/Lactic Acid/Malonic Acid System

“…The improvement of the stability of transaminase often requires the assistance of molecular docking technology to find the rigid region and flexible region of its protein structure . In addition, excellent results have been achieved by consensus mutagenesis strategies to alter the thermostability of enzymes . The sequences of 50 AT -ATA homologues (Table S4) were compared by multiple sequence alignment (Figure S2).…”

“…24 In addition, excellent results have been achieved by consensus mutagenesis strategies to alter the thermostability of enzymes. 34 The sequences of 50 AT-ATA homologues (Table S4) were compared by multiple sequence alignment (Figure S2). Positions with a conservation threshold over 60% were presumed as mutation sites.…”