3IIO

Evolutionary optimization of computationally designed enzymes: Kemp eliminases of the KE07 series

Summary for 3IIO

• Entry DOI: 10.2210/pdb3iio/pdb
• Related: 3IIP 3IIV
• Descriptor: KE07 (2 entities in total)
• Functional Keywords: beta barrel, lyase
• Biological source: Escherichia coli
• Total number of polymer chains: 6
• Total formula weight: 174324.88

Authors

Khersonsky, O.,Dym, O.,Tawfik, D.S. (deposition date: 2009-08-03, release date: 2010-03-02, Last modification date: 2023-11-01)

Primary citation

Khersonsky, O.,Rothlisberger, D.,Dym, O.,Albeck, S.,Jackson, C.J.,Baker, D.,Tawfik, D.S.
Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series.
J.Mol.Biol., 396:1025-1042, 2010

PubMed Abstract: Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k(cat)/K(M) value of approximately 2600 s(-1) M(-1) and an approximately 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.

PubMed: 20036254
DOI: 10.1016/j.jmb.2009.12.031

Experimental method

X-RAY DIFFRACTION (2.25 Å)