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
ID: NFhd35s43
Submitter: niayesh zarifi
Submission Date: May 5, 2020, 6:22 a.m.
Version: 1
Number of data points | 12 |
Proteins | Kemp Eliminase HG3.17 ; Kemp Eliminase HG4 ; Kemp Eliminase HG3.14 ; Kemp Eliminase HG3.7 ; Kemp Eliminase HG3.3b ; Kemp Eliminase HG3 |
Unique complexes | 6 |
Assays/Quantities/Protocols | Experimental Assay: Steady-state kinetics ; Derived Quantity: SD of Steady-state kinetics |
Libraries | Catalytic efficiency measured in kcat/Km for all the kemp eliminases of the HG series |
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
---|
Structure ID | Release Date | Resolution | Structure Title |
---|---|---|---|
1GOK | 2001-10-22T00:00:00+0000 | 1.14 | Thermostable xylanase I from Thermoascus aurantiacus- Crystal form II |
1GOM | 2001-10-22T00:00:00+0000 | 1.92 | Thermostable xylanase I from Thermoascus aurantiacus- Crystal form I |
1GOO | 2001-10-22T00:00:00+0000 | 1.87 | Thermostable xylanase I from Thermoascus aurantiacus - Cryocooled glycerol complex |
1GOQ | 2001-10-23T00:00:00+0000 | 1.8 | Thermostable xylanase I from Thermoascus aurantiacus - Room temperature xylobiose complex |
1GOR | 2001-10-23T00:00:00+0000 | 1.7 | THERMOSTABLE XYLANASE I FROM THERMOASCUS AURANTIACUS - XYLOBIOSE COMPLEX AT 100 K |
1I1W | 2001-02-04T00:00:00+0000 | 0.89 | 0.89A Ultra high resolution structure of a Thermostable Xylanase from Thermoascus Aurantiacus |
1I1X | 2001-02-04T00:00:00+0000 | 1.11 | 1.11 A ATOMIC 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 |
1TUX | 1998-10-29T00:00:00+0000 | 1.8 | HIGH RESOLUTION CRYSTAL STRUCTURE OF A THERMOSTABLE XYLANASE FROM THERMOASCUS AURANTIACUS |
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. |
Percent Identity | Matching Chains | Protein | Accession | Entry Name |
---|---|---|---|---|
96.0 | Kemp Eliminase HG3 | P23360 | XYNA_THEAU |