Exploring the challenges of computational enzyme design by rebuilding the active site of a dehalogenase

Abstract

Rational enzyme design presents a major challenge that has not been overcome by computational approaches. One of the key challenges is the difficulty in assessing the magnitude of the maximum possible catalytic activity. In an attempt to overcome this challenge, we introduce a strategy that takes an active enzyme (assuming that its activity is close to the maximum possible activity), design mutations that reduce the catalytic activity, and then try to restore that catalysis by mutating other residues. Here we take as a test case the enzyme haloalkane dehalogenase (DhlA), with a 1,2-dichloroethane substrate. We start by demonstrating our ability to reproduce the results of single mutations. Next, we design mutations that reduce the enzyme activity and finally design double mutations that are aimed at restoring the activity. Using the computational predictions as a guide, we conduct an experimental study that confirms our prediction in one case and leads to inconclusive results in another case with 1,2-dichloroethane as substrate. Interestingly, one of our predicted double mutants catalyzes dehalogenation of 1,2-dibromoethane more efficiently than the wild-type enzyme.

Development of a versatile and efficient C–N lyase platform for asymmetric hydroamination via computational enzyme redesign

“…It is an arduous task that does not involve knowledge of what determines new activity, and the quality of the said starting point is unknown since iterative rounds of mutagenesis target sites scattered throughout the global protein structure 37 . An alternative avenue in engineering existing enzymes has proven successful in enhancing our ability to recognize the origin of enzymes' remarkable performances using theoretical and computational methods [38][39][40][41][42][43] . By exploring a small fraction of the vast sequence space, successful examples of studies focused on reshaping active sites that accommodate individual substrates have been reported [44][45][46][47] .…”

Enhancing ade novoenzyme activity by computationally-focused ultra-low-throughput screening

“…48,49,[91][92][93][94][95] In addition, whereas we and others have been able to obtain high delity with experimental values across a wide range of enzymes and enzyme variants even in the case of far more complex systems than the current Kemp eliminase. [98][99][100][104][105][106][107][108][109] This makes EVB useful as a predictive tool for systems where the changes in energy involved are not as subtle as in the case of Kemp elimination.…”

Development of a versatile and efficient C-N lyase platform for asymmetric hydroamination via computational enzyme redesign

“…It is an arduous task that does not involve knowledge of what determines new activity, and the quality of the said starting point is unknown since iterative rounds of mutagenesis target sites scattered throughout the global protein structure 37 . An alternative avenue in engineering existing enzymes has proven successful in enhancing our ability to recognize the origin of enzymes' remarkable performances using theoretical and computational methods [38][39][40][41][42][43] . By exploring a small fraction of the vast sequence space, successful examples of studies focused on reshaping active sites that accommodate individual substrates have been reported [44][45][46][47] .…”