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Constrained evolution of a bispecific enzyme: lessons for biocatalyst design

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Correction: Copper(ii) catalyzed synthesis of novel helical luminescent benzo[4,5]imidazo[1,2-a][1,10]phenanthrolines via an intramolecular C–H amination reaction

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Organic & Biomolecular Chemistry Vol - 15 Issue - 3

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Organic & Biomolecular Chemistry Vol -2

Constrained evolution of a bispecific enzyme: lessons for biocatalyst design

Content Provider	Royal Society of Chemistry (RSC)
Author	Sugrue, E. Jackson, C. J. Scott, C.
Copyright Year	2017
Abstract	One of the central goals of protein design and engineering is to be able to accurately predict the effects of a mutation on stability and activity. However, the genetic context into which mutations are introduced can lead to complex interactions between the mutation and other amino acids and unpredictable, non-additive, effects. This phenomenon is known as intramolecular epistasis and has been shown to restrict evolutionary paths through laboratory directed evolution experiments and ancestral protein reconstruction, but has rarely been studied at a quantitative level in naturally evolving enzymes. Atrazine-specific and atrazine/ametryn bispecific triazine hydrolases (TrzN) have evolved in different bacterial strains over the past fifty years in response to the presence of the synthetic herbicides atrazine and ametryn. Here, we have investigated all 24 evolutionary trajectories that are possible from monofunctional to bispecific TrzN isoforms in terms of activity, stability, expression and structure. The results reveal that half of these trajectories are unviable due to inactive intermediates, with only 1/24 trajectories exhibiting consistent improvement in bispecificity. The most viable path requires the mutation of Gln241 to Glu241 first, which increases activity 3-fold with atrazine and 105-fold with ametryn, which is further optimised in subsequent evolutionary steps. The epistatic interactions between mutations, involving control of the pKa of catalytic residues, the thermostability of the protein, and soluble expression are shown to be responsible for the bottlenecks in this evolutionary landscape. This comprehensive analysis of the evolution of bispecificity highlights the importance of epistasis in protein engineering and evolution, which makes identifying the correct sequence in which to combine mutations extremely important.
Starting Page	937
Ending Page	946
Page Count	10
File Format	HTM / HTML PDF
ISSN	14770520
Volume Number	15
Issue Number	4
Journal	Organic & Biomolecular Chemistry
DOI	10.1039/c6ob02355j
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Protein primary structure Thermostability Protein engineering Directed evolution Acid dissociation constant Triazine Epistasis Mutation Atrazine
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Biochemistry Physical and Theoretical Chemistry

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