Engineering a Catalytically Efficient Recombinant Protein Ligase.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Peptidyl bond formation is crucial in protein chemistry, but fast-acting ligases are rare compared to proteases.
• The enzyme OaAEP1 from Oldenlandia affinis exhibits weak peptide cyclase activity despite a fold similar to asparaginyl endopeptidases (AEPs).

Purpose of the Study:

• To determine the atomic structure of OaAEP1 in its preactivation form.
• To elucidate the enzyme's activation mechanism and structural features critical for ligation.
• To engineer a highly active ligase for protein modification applications.

Main Methods:

• Determined the atomic structure of OaAEP1 using X-ray crystallography at 2.56 Å resolution.
• Performed biochemical analyses to understand enzyme activation and ligation.
• Utilized structure-based mutagenesis to engineer enhanced catalytic activity.

Main Results:

• The first atomic structure of preactivation OaAEP1 was solved, revealing its activation mechanism.
• Structure-based engineering yielded an ultrafast variant with hundreds of times faster catalytic kinetics.
• The engineered variant efficiently ligates well-folded protein substrates at submicromolar enzyme concentrations.

Conclusions:

• Structure-based engineering of OaAEP1 significantly enhances its ligation activity.
• The developed ultrafast ligase is a novel recombinant tool for challenging protein labeling and modifications.
• This work overcomes previous limitations in enzyme-catalyzed protein ligation.