Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes

Area of Science:

• Enzyme Engineering
• Structural Biology
• Biocatalysis

Background:

• Directed evolution of E. coli transketolase (TKT) using smart libraries progressively altered substrate specificity.
• Previous in silico modeling of TKT variants had limitations due to accumulated errors with successive mutations.
• Understanding the structural basis of altered substrate binding is crucial for further enzyme engineering.

Purpose of the Study:

• To determine the crystal structure of the S385Y/D469T/R520Q E. coli transketolase variant.
• To investigate the molecular interactions governing the binding of three aromatic aldehyde substrates.
• To validate the hypothesis of divergent binding modes in an evolutionary intermediate enzyme variant.

Main Methods:

• X-ray crystallography was employed to obtain the high-resolution structure of the evolved TKT variant.
• Molecular docking simulations were performed using three distinct benzaldehyde derivatives as substrates.
• Kinetic evaluations were conducted to assess substrate binding affinities and preferences.

Main Results:

• The crystal structure revealed a modified active site with two distinct binding pockets, separated by the D469T mutation.
• These pockets facilitate differential binding of aromatic aldehydes, with 3-formylbenzoic acid (3-FBA) and 4-FBA showing pocket preference.
• The less accepted substrate, 3-hydroxybenzaldehyde (3-HBA), exhibited intermediate binding affinity across both pockets.

Conclusions:

• The crystal structure of the evolved TKT variant supports the hypothesis of divergent substrate binding modes.
• The identified active site architecture, featuring two π-π stacking-supporting pockets, explains the altered substrate specificity.
• Obtaining crystal structures of evolved enzyme variants is vital for reliable and continued application of smart library design strategies.