Figure 1
From: Dissecting the evolvability landscape of the CalB active site toward aromatic substrates
Schematic representation of the Iterative Semi-Rational Design (ISRD) strategy employed in the present work. (A) Identification and selection of active-site mutational ‘hot spots’ by convergence of two approaches. First, identification of residue positions that stabilize the ligand-enzyme complex is achieved by virtual docking, after which involvement of those positions in the maintenance of structure and topology of the active-site cavity is performed by a Residue Interaction Network (RIN) analysis12. Enzyme-substrate complex formation is accomplished by mimicking an induced fit mechanism using virtual docking. Targeted active-site positions are selected based on ranking of energy contributions for each docking complex and residue interaction likelihood with the desired substrates. RIN centrality measures provide information on residue ‘hot spots’ that are more likely to maintain CalB structure. As a result, positions with low betweenness scores are prioritized, i.e. positions with lower impact on protein cavity disruption. (B) Building and screening of the mutant libraries using a vinyl analog to measure the synthetic activity of CalB in organic solvent conditions (see Experimental Procedures for details). Genetic identification of the most active mutants allows selection of the best candidates as template(s) for the next round of mutational selection. (C) Modeling of experimentally selected variants with favorable active-site replacements as template for the next round of virtual docking (A). This complementary procedure combining experimental and computational approaches is iteratively repeated to improve esterification activity catalyzed by CalB.
