Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis

Sorbicillinoids are yellow secondary metabolites synthesized
through
an elegant combination of enzymatic and spontaneous biochemical processes.
The flavin-dependent monooxygenase SorC and oxidase SorD are crucial
in this interplay, enabling the generation of a diverse array of functionally
complex sorbicillinoids. By solving the crystal structures of SorC
and SorD from Penicillium chrysogenum with sorbicillin bound in the active site, we describe the catalytically
active binding conformations, crucial for attaining enantioselective
and stereoselective control in these enzymatic reactions. The structure
of SorC was resolved with the cofactor FAD in its out state, which allowed us to identify key residues that modulate flavin
mobility and other conformational changes. Catalytic residues of SorC
were also confirmed by detailed characterization of wild-type and
several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome
integration system, we could heterologously express the flavin-dependent
oxidase SorD from P. chrysogenum in Aspergillus niger with high yields and purity. This
allowed us to obtain the crystal structure of SorD with sorbicillin
bound in a viable catalytic conformation. Structural analysis of the
obtained complex provided insights into the substrate binding pose
and highlighted potentially critical active site residues. Ultimately,
having both SorC and SorD at our disposal enabled us to investigate
their functions and interplays in the biosynthesis of a vast array
of functionally complex sorbicillinoids.