Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst


The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we used room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 160 M−1s−1). We observed that catalytic residues were increasingly rigidified, the active site became better pre-organized, and its entrance was widened. Based on these observations, we engineered HG4, an efficient biocatalyst (kcat/KM 120,000 M−1s−1) containing active-site mutations found during evolution but not distal ones. HG4 structures revealed that its active site was pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data

Submission Details

ID: NFhd35s43

Submitter: niayesh zarifi

Submission Date: May 5, 2020, 6:22 a.m.

Version: 1

Publication Details
1Aron Broom, 1Rojo V. Rakotoharisoa, 2Michael C. Thompson, 1Niayesh Zarifi, 1Erin Nguyen, 1Nurzhan Mukhametzhanov, 2Lin Liu, 2James S. Fraser, 1Roberto A. Chica (2020) Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst; unpublished work from 1Department of Chemistry and Biomolecular Sciences, University of Ottawa, 2Department of Bioengineering and Therapeutic Science, University of California, San Francisco Year: 2020 group
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3O2L 2010-07-22T00:00:00+0000 2.0 Crystal Structure of an Inactive Kemp Elimination Design HG-1
1I1W 2001-02-04T00:00:00+0000 0.89 0.89A Ultra high resolution structure of a Thermostable Xylanase from Thermoascus Aurantiacus
1K6A 2001-10-15T00:00:00+0000 1.14 Structural studies on the mobility in the active site of the Thermoascus aurantiacus xylanase I
2BNJ 2005-03-25T00:00:00+0000 1.6 The xylanase TA from Thermoascus aurantiacus utilizes arabinose decorations of xylan as significant substrate specificity determinants.
3NYD 2010-07-14T00:00:00+0000 1.23 Crystal Structure of Kemp Eliminase HG-2 Complexed with Transition State Analog 5-Nitro Benzotriazole
7K4Z 2020-09-16T00:00:00+0000 1.08 Crystal structure of Kemp Eliminase HG3.17 in complex with the transition state analog 6-nitrobenzotriazole
7K4W 2020-09-16T00:00:00+0000 1.9 Crystal structure of Kemp Eliminase HG3.17 in the inactive state
7K4S 2020-09-16T00:00:00+0000 2.0 Crystal structure of Kemp Eliminase HG3.7
7K4P 2020-09-16T00:00:00+0000 1.08 Crystal structure of Kemp Eliminase HG3

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.0 Kemp Eliminase HG3 P23360 XYNA_THEAU