Highly active enzymes by automated combinatorial backbone assembly and sequence design.

Nature Communications
Gideon LapidothSarel J Fleishman

Abstract

Automated design of enzymes with wild-type-like catalytic properties has been a long-standing but elusive goal. Here, we present a general, automated method for enzyme design through combinatorial backbone assembly. Starting from a set of homologous yet structurally diverse enzyme structures, the method assembles new backbone combinations and uses Rosetta to optimize the amino acid sequence, while conserving key catalytic residues. We apply this method to two unrelated enzyme families with TIM-barrel folds, glycoside hydrolase 10 (GH10) xylanases and phosphotriesterase-like lactonases (PLLs), designing 43 and 34 proteins, respectively. Twenty-one GH10 and seven PLL designs are active, including designs derived from templates with <25% sequence identity. Moreover, four designs are as active as natural enzymes in these families. Atomic accuracy in a high-activity GH10 design is further confirmed by crystallographic analysis. Thus, combinatorial-backbone assembly and design may be used to generate stable, active, and structurally diverse enzymes with altered selectivity or activity.

References

Feb 15, 1990·Archives of Biochemistry and Biophysics·D P DumasJ R Wild
Sep 14, 1986·Biochemical and Biophysical Research Communications·S G WithersN R Gilkes
Oct 25, 1994·Proceedings of the National Academy of Sciences of the United States of America·W P Stemmer
Apr 9, 1999·Biotechnology Progress·P Bajpai
Jan 3, 2001·Nature Structural Biology·B HöckerR Sterner
May 1, 2001·Nature Biotechnology·V SieberF H Arnold
May 1, 1997·Acta Crystallographica. Section D, Biological Crystallography·G N MurshudovE J Dodson
Nov 13, 2004·Proceedings of the National Academy of Sciences of the United States of America·Birte HöckerReinhard Sterner
Dec 2, 2004·Acta Crystallographica. Section D, Biological Crystallography·Paul Emsley, Kevin Cowtan
Jun 16, 2005·Annual Review of Biochemistry·Christine A Orengo, Janet M Thornton
Nov 10, 2005·Chemical Reviews·Reinhard Sterner, Birte Höcker
Dec 6, 2005·Chembiochem : a European Journal of Chemical Biology·Olga Khersonsky, Dan S Tawfik
Dec 22, 2005·Acta Crystallographica. Section D, Biological Crystallography·Philip Evans
Sep 19, 2006·Journal of Molecular Biology·Lutz Riechmann, Greg Winter
Nov 9, 2006·Protein Engineering, Design & Selection : PEDS·Michelle M MeyerFrances H Arnold
Dec 14, 2006·Acta Crystallographica. Section D, Biological Crystallography·Airlie J McCoy
Mar 8, 2008·Science·Lin JiangDavid Baker
Mar 21, 2008·Nature·Daniela RöthlisbergerDavid Baker
May 20, 2008·Journal of Molecular Biology·Mikael EliasEric Chabriere
Jun 11, 2008·Methods in Molecular Biology·Yoav Peleg, Tamar Unger
Jul 18, 2008·Proceedings of the National Academy of Sciences of the United States of America·Tanmay A M BharatBirte Höcker
Aug 5, 2008·Journal of Molecular Biology·Satoshi Akanuma, Akihiko Yamagishi
Feb 25, 2009·Proceedings of the National Academy of Sciences of the United States of America·Jörg ClarenReinhard Sterner
Mar 25, 2009·Proceedings of the National Academy of Sciences of the United States of America·Pete HeinzelmanFrances H Arnold
May 28, 2009·Proceedings of the National Academy of Sciences of the United States of America·Paul M MurphyDavid Baker
Nov 26, 2009·Nature Reviews. Molecular Cell Biology·Philip A Romero, Frances H Arnold
Dec 17, 2009·BMC Bioinformatics·Christiam CamachoThomas L Madden
Feb 18, 2009·Global Change Biology. Bioenergy·Dylan Dodd, Isaac K O Cann
Aug 19, 2010·Protein Science : a Publication of the Protein Society·David Baker
Oct 29, 2010·The Journal of Biological Chemistry·Jeng Yeong ChowWen Shan Yew
Feb 15, 2012·Journal of the American Chemical Society·Simone EisenbeisBirte Höcker
Mar 8, 2012·PLoS Computational Biology·Nicholas FurnhamJanet M Thornton
Apr 17, 2012·Acta Crystallographica. Section D, Biological Crystallography·Pavel V AfoninePaul D Adams
Jun 12, 2012·Proceedings of the National Academy of Sciences of the United States of America·Olga KhersonskyDan S Tawfik
Nov 10, 2012·Scientific Reports·Julien HiblotMikael Elias
Feb 7, 2013·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Linda ReinhardManfred S Weiss
Mar 26, 2013·Angewandte Chemie·Gert KissK N Houk

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Citations

Aug 17, 2019·Nature Reviews. Molecular Cell Biology·Brian Kuhlman, Philip Bradley
Aug 11, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Lucas Ferreira RibeiroMaría-Eugenia Guazzaroni
Aug 24, 2019·PLoS Computational Biology·Shira WarszawskiSarel J Fleishman
Aug 29, 2019·PLoS Computational Biology·Jonathan Yaacov WeinsteinSarel Jacob Fleishman
Mar 4, 2020·Scientific Reports·Matias Romero VictoricaPaola M Talia
Jun 3, 2020·Nature Methods·Julia Koehler LemanRichard Bonneau
Oct 12, 2020·Protein Science : a Publication of the Protein Society·Rosalie Lipsh-SokolikSarel J Fleishman
Jan 17, 2021·Current Opinion in Structural Biology·Sergio Romero-RomeroBirte Höcker
Nov 19, 2020·Proceedings of the National Academy of Sciences of the United States of America·Shane J CaldwellCathleen Zeymer
Oct 3, 2018·Molecular Cell·Olga KhersonskySarel J Fleishman
Mar 31, 2021·Journal of Molecular Biology·Yoav PelegSarel J Fleishman
Jul 3, 2021·Frontiers in Bioengineering and Biotechnology·Marc SchererElena Bencurova
Aug 29, 2020·Structure·Ratul ChowdhuryCostas D Maranas
Jul 25, 2019·Current Opinion in Chemical Biology·William M DawsonDerek N Woolfson
Jul 5, 2021·Biotechnology Advances·Lunjie WuYi-Lei Zhao

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Methods Mentioned

BETA
affinity-purification
protein folding
gel filtration
PCR
X-ray

Software Mentioned

RosettaScripts
BLASTP
Mosflm
AddGene
PHASER
COOT
Rosetta
RosettaDesign
Prism
REFMAC5

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