Unexpected assembly machinery for 4(3H)-quinazolinone scaffold synthesis

Area of Science:

• Biochemistry
• Natural Product Biosynthesis
• Enzymology

Background:

• 4(3H)-quinazolinone is a crucial scaffold in natural products and drugs.
• Fungal enzymatic synthesis mechanisms, particularly for nitrogen origins, remain underexplored beyond fumiquinazolines.

Purpose of the Study:

• To elucidate the enzymatic mechanism for 4(3H)-quinazolinone biosynthesis in the chrysogine pathway.
• To identify the origins of nitrogen atoms (N-1 and N-3) in the fungal 4(3H)-quinazolinone scaffold.
• To characterize the enzymes involved in this novel pathway.

Main Methods:

• Biochemical investigation of the chrysogine pathway.
• Characterization of a fungal two-module nonribosomal peptide synthase (ftChyA).
• Enzymatic assays using an α-ketoglutarate-dependent dioxygenase (ftChyM).

Main Results:

• A novel 4(3H)-quinazolinone assembly machinery involving ftChyA with an unusual condensation domain was identified.
• The N-3 atom originates from inorganic ammonium ions or L-glutamine (L-Gln).
• ftChyM catalyzes oxidative cleavage of a tripeptide to a dipeptide, revealing an alternative synthesis route.

Conclusions:

• Uncovered a unique nonribosomal peptide release and tailoring mechanism.
• Demonstrated an alternative enzymatic route for synthesizing 4(3H)-quinazolinone scaffolds in fungi.
• Expanded understanding of nitrogen incorporation in fungal secondary metabolite biosynthesis.