PDBsum entry 3nq2

Lyase

PDB id: 3nq2

Contents

Protein chains

247 a.a. *

Ligands

IMD ×2

Waters ×157

* Residue conservation analysis

PDB id:

3nq2

Name:

Lyase

Title:

Optimization of the in silico designed kemp eliminase ke70 by computational design and directed evolution r2 3/5g

Structure:

Deoxyribose phosphate aldolase. Chain: a, b. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.02Å R-factor: 0.208 R-free: 0.260

Authors:

O.Khersonsky,D.Rothlisberge,A.M.Wollacott,O.Dym,D.Baker,D.S.Tawfik, Israel Structural Proteomics Center (Ispc)

Key ref:

O.Khersonsky et al. (2011). Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution. J Mol Biol, 407, 391-412. PubMed id: 21277311

Date:

29-Jun-10 Release date: 09-Feb-11

Protein chains

No UniProt id for this chain

Struc: 247 a.a.

J Mol Biol 407:391-412 (2011)

PubMed id: 21277311

Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution.

O.Khersonsky, D.Röthlisberger, A.M.Wollacott, P.Murphy, O.Dym, S.Albeck, G.Kiss, K.N.Houk, D.Baker, D.S.Tawfik.

ABSTRACT

Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5×10(4) s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis.