Structural insights into CYP107G1 from rapamycin-producing Streptomyces rapamycinicus

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Rapamycin, an immunosuppressant and antiproliferative macrolide, is produced by Streptomyces rapamycinicus.
• Two cytochrome P450 enzymes, CYP107G1 and CYP122A2, are essential for rapamycin biosynthesis, catalyzing specific oxidation reactions.

Purpose of the Study:

• To elucidate the structural and biochemical characteristics of CYP107G1 and CYP122A2 involved in rapamycin biosynthesis.
• To understand the substrate-binding mechanisms of these P450 enzymes.

Main Methods:

• Cloning and recombinant expression of CYP107G1 and CYP122A2 genes in Escherichia coli.
• Biochemical characterization including spectral analysis and substrate binding assays (rapamycin titration).
• X-ray crystallography to determine the structures of CYP107G1 and its complex with everolimus.

Main Results:

• Both CYP107G1 and CYP122A2 exhibited low spin states and bound rapamycin with high affinity (K_d values in the low μM range).
• The crystal structure of CYP107G1 revealed a canonical P450 scaffold with a large, solvent-exposed substrate pocket.
• The CYP107G1-everolimus complex structure showed the drug bound in a unique conformation within a rigid pocket, indicating adaptability through flexible loops.

Conclusions:

• CYP107G1 possesses a structurally stable yet adaptable active site capable of accommodating bulky macrolide substrates like everolimus.
• These findings provide insights into the enzymatic mechanisms underlying rapamycin biosynthesis and potential for drug development.