TY - JOUR

T1 - Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase

AU - Cea-Rama, Isabel

AU - Coscolín, Cristina

AU - Katsonis, Pangiotis

AU - Bargiela, Rafael

AU - Golyshin, Peter

AU - Lichtarge, Olivier

AU - Ferrer, Manuel

AU - Sanz-Aparicio, Julia

N1 - © 2021 The Author(s).

PY - 2021

Y1 - 2021

N2 - Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH3) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.

AB - Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH3) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.

KW - Crystal structure

KW - EA, evolutionary action

KW - ET, evolutionary trace

KW - Eapp, apparent enantioselectivity

KW - Esterase

KW - Evolutionary trace

KW - HEPES, 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

KW - Ni-NTA, nickel-nitrilotriacetic acid

KW - Promiscuity

KW - Protein engineering

KW - Specificity

U2 - 10.1016/j.csbj.2021.04.041

DO - 10.1016/j.csbj.2021.04.041

M3 - Article

C2 - 33995922

VL - 19

SP - 2307

EP - 2317

JO - Computational and Structural Biotechnology Journal

JF - Computational and Structural Biotechnology Journal

SN - 2001-0370

ER -