Abstract
Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.
Highlights
The distribution of amino acids in multiple sequence alignments (MSAs) of homologous proteins reveals the most conserved positions in the different sequences (Lehmann et al, 2000; Lehmann and Wyss, 2001)
The departure point of this study was the OB-1 mutant, a HRPL evolved from the basidiomycete PM1 laccase (PM1Lac) (Coll et al, 1993; Mate et al, 2010)
OB-1 carries the V[α10]D-N[α23]K-A[α87]T-V162A-H208Y-S224G-A239PD281E-S426N-A461T mutations that improve its secretion and activity in S. cerevisiae
Summary
The distribution of amino acids in multiple sequence alignments (MSAs) of homologous proteins reveals the most conserved positions in the different sequences (Lehmann et al, 2000; Lehmann and Wyss, 2001). Organized into three cupredoxin domains, laccase catalysis is governed by the T1Cu site where the substrate binds, and the T2/T3 trinuclear copper cluster located 12 Å away that is involved in the conversion of molecular oxygen to water (Jones and Solomon, 2015) In this context, high-redox potential laccases (HRPLs) from white-rot fungi are of particular interest as they have outstanding oxidative capabilities that are dependent on both a high-redox potential at the T1Cu site and a relaxed substrate binding mode (Rodgers et al, 2010; Mate and Alcalde, 2015; Mateljak et al, 2019a). The most promising mutations were subjected to site-directed recombination in vivo to search for positive epistatic combinations, and the final mutant was characterized biochemically
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