Molecular Basis for Asynchronous Chain Elongation During Rifamycin Antibiotic Biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Rifamycin synthetase (RIFS) is a massive enzyme complex essential for rifamycin B antibiotic production.
• RIFS functions as a hybrid enzymatic assembly line, combining nonribosomal peptide synthetase and polyketide synthase modules.
• Detailed structural and biochemical data on purified RIFS components are limited.

Purpose of the Study:

• To elucidate the biosynthetic mechanism of RIFS by analyzing its first polyketide synthase module (M1).
• To investigate the structural and dynamic interactions within the M1 module during polyketide chain elongation.
• To understand how protein architecture influences enzymatic activity in megasynthase systems.

Main Methods:

• Thiol-selective crosslinking to probe protein-protein interactions.
• Cryogenic electron microscopy (cryo-EM) for high-resolution structural analysis.
• Single-turnover kinetic assays to measure reaction rates and intermediate formation.

Main Results:

• Crosslinking data revealed conformational asymmetry in ketosynthase (KS)-substrate carrier protein (CP) interactions.
• Cryo-EM unveiled the unique architecture of the M1 module, supporting domain co-migration.
• Removal of C-terminal dimeric interfaces increased KS-CP interactions but not product formation, suggesting a regulatory role.

Conclusions:

• The C-terminal dimeric interfaces in bacterial polyketide synthases likely orchestrate CP domain movement between KS active sites.
• Understanding these molecular details is crucial for engineering novel biosynthetic enzymes.
• This research provides insights into natural antibiotic biosynthesis and the design of artificial megasynthases.