Highly active enzymes by automated combinatorial backbone assembly and sequence design

By Gideon Lapidoth, O. Khersonsky, R. Lipsh, Orly Dym, Shira Albeck, S. Rogotner, Sarel Jacob Fleishman. 2018

47 citations recorded by the source corpus.

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. Computationally designed enzymes often show lower activity or stability than their natural counterparts. Here, the authors present an evolution-inspired method for automated enzyme design, creating stable enzymes with accurate active site architectures and wild-type-like activities.

Subfields: Materials Chemistry, Molecular Biology

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Materials Chemistry

1. Che Yang
2. S. Rosset
3. Bruno E. Correia
4. Sarel Jacob Fleishman

In Molecular Biology

1. Bruno E. Correia
2. S. Rosset
3. D. Listov
4. Che Yang
5. Rosalie Lipsh-Sokolik
6. Sarel Jacob Fleishman

Citations in the corpus

• Assessing and enhancing foldability in designed proteins (2021, methodology): “Our strategy is based on recent methods developed in our lab to design new backbones through the modular assembly of large (60–150 amino acid) backbone fragments of natural enzymes.(25,30,31) Modular assembly and design was successful in generating accurate antibodies(27) and ultrahigh specificity b”
• Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion (2020, unknown): “The de novo design of catalytic functions, however, has been challenged in part by the difficulty in programming flexible loops; the most successful approaches to date have reused loops found in nature (14, 15).”