Header

UZH-Logo

Maintenance Infos

Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase


Obexer, Richard; Godina, Alexei; Garrabou, Xavier; Mittl, Peer R E; Baker, David; Griffiths, Andrew D; Hilvert, Donald (2017). Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase. Nature Chemistry, 9(1):50-56.

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 >10(9) rate 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.

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 >10(9) rate 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.

Statistics

Citations

7 citations in Web of Science®
10 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

0 downloads since deposited on 23 Jun 2017
0 downloads since 12 months

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:January 2017
Deposited On:23 Jun 2017 10:19
Last Modified:24 Jun 2017 07:54
Publisher:Nature Publishing Group
ISSN:1755-4330
Publisher DOI:https://doi.org/10.1038/nchem.2596
PubMed ID:27995916

Download

Preview Icon on Download
Content: Published Version
Filetype: PDF - Registered users only
Size: 5MB
View at publisher