In Silico Prediction Methods for Site-Saturation Mutagenesis

Abstract

Directed enzyme evolution has proven to be a powerful means to endow biocatalysts with novel catalytic repertoires. Apart from completely random gene mutagenesis, site-directed or site-saturation mutagenesis requires a semi-rational selection of the amino acid positions or the substituted residues, which can dramatically reduce the screening efforts in protein engineering. To this end, in silico prediction methods play a pivotal role in targeting site-saturation mutagenesis. In this chapter, we provide two distinct computational methods, (a) conformational dynamics-guided design and (b) protein-ligand interaction fingerprinting analysis, to identify specific positions for site-saturation mutagenesis toward manipulating substrate specificity/stereoselectivity of an alcohol dehydrogenase, and improving activity of a carboxylic acid reductase, respectively.

Keywords: Alcohol dehydrogenase; Carboxylic acid reductase; Conformational dynamics; Enzyme engineering; In silico; Protein–ligand interaction; Rational design; Site-specific saturation mutagenesis.