Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments

Abstract

Error-prone PCR, DNA shuffling, and saturation mutagenesis are techniques used by protein engineers to mimic the natural "evolutionary walk" that conjures new enzymes. Rational design is often critical in efforts to accelerate this "random walk" into a "resolute sprint." Previous work by our group established a computational method for detecting active sites (CLASP) based on spatial and electrostatic properties of catalytic residues, and a method to quantify promiscuous activities in a wide range of proteins (PROMISE). Here, we describe a rational design flow (DECAAF) based on the PROMISE methodology to choose a protein which, when subjected to minimal mutations, is most likely to mirror the scaffold of a desired enzymatic function. Modeling the diversity in catalytic sites and providing precise user control to guide the search is a key goal of our implementation. The flow details have been worked out in a real-life example to select a plant protein to substitute for human neutrophil elastase in a chimeric antimicrobial enzyme designed to bolster the innate immune defense system in plants.

An overview of the alpha-, beta- and gamma-carbonic anhydrases fromBacteria: can bacterial carbonic anhydrases shed new light on evolution of bacteria?

“…The hypothesis presented above is the result of the detailed phylogenetic analysis performed by us, according to which from the ancestral Ur-CA, the g-class has arisen first, followed by the b-class, whereas the a-class CAs was the last originated CA class. This hypothesis might be corroborated also by considering the theory of ''enzyme promiscuity'' [67][68][69] . Catalytic sites can undergo structural changes while the function of the enzyme remains almost the same 70 .…”

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

“…The theoretical foundation of CLASP is that the electrostatic potential difference (EPD) in cognate pairs of active site residues is conserved in proteins with the same functionality. The significance of EPD was extended to a method for enumerating possible pathways for proton abstraction in the active site [57], compute electrostatic perturbations induced by ligand binding [58], and propose a rational design-flow for directed evolution [59], [60]. Recently, we proposed a methodology for the multiple sequence alignment of related proteins with known structures using electrostatic properties as an additional discriminator and identified mutations that might be the source of functional divergence in a protein family.…”

Biological messiness vs. biological genius: Mechanistic aspects and roles of protein promiscuity

“…This method essentially identifies structurally related active sites that are represented in separate regions of ‘EC space.’ The combination of the EC phylogeny with structural considerations has nice predictive features for in silico directed evolution from promiscuous protein scaffolds [59]. However, this approach obviously requires a crystal structure to assign a promiscuity score for any enzyme, or strictly speaking its mutants.…”