Reverse evolution leads to genotypic incompatibility despite functional and active site convergence.
Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (&gt;10(4)-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks.
Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.
Miriam
M
Kaltenbach
Research School of Chemistry, Australian National University, Canberra, Australia.
Colin J
CJ
Jackson
Research School of Chemistry, Australian National University, Canberra, Australia.
Eleanor C
EC
Campbell
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Florian
F
Hollfelder
Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.
Nobuhiko
N
Tokuriki
D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D005838:Genotype; D009154:Mutation; D010641:Phenotype; D044345:Phosphoric Triester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D012641:Selection, Genetic
D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D044345:Phosphoric Triester Hydrolases
E. coli; biochemistry; enzyme evolution; evolutionary biology; evolutionary reversibility; genomics; laboratory evolution; mutational epistasis; sequence incompatibility; sequence-structure-function relationship