Structural basis for differentiation between two classes of thiolase: Degradative vs biosynthetic thiolase

Area of Science:

• Biochemistry and Structural Biology
• Enzymology
• Bioinformatics

Background:

• Thiolases are enzymes with two categories: degradative and biosynthetic, catalyzing reactions in opposite directions.
• Both thiolase classes share a catalytic triad but differ in substrate chain length acceptance.
• No clear structural or residue patterns have been identified to distinguish between degradative and biosynthetic thiolases.

Purpose of the Study:

• To elucidate structural and functional features distinguishing degradative from biosynthetic thiolases.
• To utilize *Pseudomonas putida* KT2440's tetrameric degradative thiolase (PcaF) as a model system.
• To identify unique characteristics of degradative thiolases through structural and bioinformatics analyses.

Main Methods:

• Structural studies, including X-ray crystallography, of the PcaF enzyme.
• Bioinformatics analyses to compare active site architectures and identify distinguishing features.
• Site-directed mutagenesis (H356A mutant) to investigate the role of specific residues in enzyme function.

Main Results:

• Degradative thiolases exhibit different active site architectures compared to biosynthetic thiolases.
• A unique covering loop, exclusive to degradative thiolases, was identified, influencing substrate binding.
• Mutation of H356 in PcaF trapped a gridlocked structure, showing covalent linkage of Coenzyme A (CoA) to the catalytic cysteine.

Conclusions:

• Degradative thiolases possess distinct structural elements, including a unique covering loop and active site architecture, that differentiate them from biosynthetic thiolases.
• Specific residues, like H356 in PcaF, play crucial roles in tethering Coenzyme A (CoA) and influencing enzyme activity.
• The identified features provide a basis for distinguishing between the two thiolase classes and understanding their functional divergence.