Functional conformations for pyruvate carboxylase during catalysis explored by cryoelectron microscopy

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Pyruvate carboxylase (PC) is a biotin-dependent enzyme catalyzing oxaloacetate production.
• PC functions via two reactions in distinct active sites, with known symmetric and asymmetric tetrameric forms.
• Understanding PC's dynamic conformations is crucial for elucidating its allosteric regulation.

Purpose of the Study:

• To investigate the conformational landscape of pyruvate carboxylase during oxaloacetate synthesis.
• To correlate specific tetrameric architectures (symmetric and asymmetric) with PC's catalytic functions.
• To characterize the structural transitions underlying PC's allosteric control.

Main Methods:

• Analysis of Staphylococcus aureus pyruvate carboxylase samples during enzymatic activity.
• Application of cryoelectron microscopy (cryo-EM) for high-resolution structural determination.
• Utilizing sorting and classification techniques to resolve different conformational states.

Main Results:

• Observed both previously defined symmetric and asymmetric PC tetrameric architectures.
• Demonstrated that PC undergoes significant conformational changes, adopting both arrangements.
• Correlated each distinct PC configuration with one of the two consecutive enzymatic reactions.

Conclusions:

• Pyruvate carboxylase transitions between symmetric and asymmetric states, coupled to its catalytic steps.
• These findings elucidate the structural dynamics governing PC's allosteric regulation.
• Cryo-EM and classification are powerful tools for studying the functional dynamics of macromolecules in action.