title: Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase.
abstract: Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >109rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland.
authors.forename: Richard
authors.initials: R
authors.lastname: Obexer
authors.affiliation: Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland.|Laboratory of Biochemistry, École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), CNRS UMR 8231, 10, rue Vauquelin, 75231 Paris Cedex 05, France.
authors.forename: Alexei
authors.initials: A
authors.lastname: Godina
authors.affiliation: Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland.
authors.forename: Xavier
authors.initials: X
authors.lastname: Garrabou
authors.affiliation: Department of Biochemistry, University of Zürich, 8057 Zürich, Switzerland.
authors.forename: Peer R E
authors.initials: PR
authors.lastname: Mittl
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Laboratory of Biochemistry, École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), CNRS UMR 8231, 10, rue Vauquelin, 75231 Paris Cedex 05, France.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Griffiths
authors.affiliation: Laboratory of Organic Chemistry, ETH Zürich, 8093 Zürich, Switzerland.
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D000595:Amino Acid Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics; D015723:Gene Library; D044085:Microfluidics / Q000379:methods*; D008958:Models, Molecular; D010957:Plasmids; D015202:Protein Engineering; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; C116609:3-hydroxybutanal; D005634:Fructose-Bisphosphate Aldolase