Focusing Mutations into the P. fluorescens Esterase Binding Site Increases Enantioselectivity More Effectively than Distant Mutations

Abstract

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

“…However, mutations closer to the catalytic core appear to have a greater effect on the selected catalytic activity (30)(31)(32)(33). These residues for mutagenesis may be selected through a random, rational or combined evolutionary approach.…”

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

“…Optimization of interactions within the binding region was expected to increase specificity. 46 The individual pocket designs ( Fig. 2) were carried out in the following sequential order:…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…For example, amino acid residues that alter substrate specificity or selectivity are commonly non-conserved residues. They are often in close contact with catalytic residues in or near the active site, cofactors or substrates Paramesvaran et al, 2009;Park et al, 2005). Another approach is the identification of sequence motifs that are thought to have been conserved during evolution (Saravanan et al, 2008).…”

“…Based on structural knowledge, it may be sufficient to target the active-site residues (Park et al, 2005;Schmitzer et al, 2004;Wilming et al, 2002;Woodyer et al, 2003). SDSM can be used to complement error-prone PCR.…”