Comprehensive characterization of ligand-induced plasticity changes in a dimeric enzyme

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Enzyme structural plasticity is key to substrate binding but challenging to study.
• AAC(6 ')-Ii, an N-acetyltransferase from Enterococcus faecium, confers aminoglycoside resistance.
• Understanding enzyme flexibility is vital for drug development.

Purpose of the Study:

• To characterize the structure of substrate-free AAC(6 ')-Ii.
• To investigate the impact of structural plasticity on enzyme function.
• To elucidate the conformational changes upon substrate binding.

Main Methods:

• X-ray crystallography to determine enzyme structure.
• Small-angle X-ray scattering (SAXS) to analyze overall structure.
• Circular dichroism (CD) spectroscopy to assess protein folding.

Main Results:

• AAC(6 ')-Ii exists in a partially unfolded state without its substrate, acetyl coenzyme A (AcCoA).
• Structural analysis revealed relative motions of dimeric enzyme components.
• Conformational changes upon AcCoA binding were identified, impacting dimerization and substrate binding.
• Flexibility of structural elements was linked to allosteric cooperativity.

Conclusions:

• AAC(6 ')-Ii exhibits significant structural plasticity, particularly in its substrate-free state.
• This flexibility is integral to its dimerization and substrate binding mechanisms.
• The findings provide structural insights into the allosteric behavior of AAC(6 ')-Ii.