Investigating and Re-Engineering Baeyer-Villiger Monooxygenase MtmOIV for Combinatorial Biosynthesis of Novel Aureolic Acid Anticancer Drug Analogues

Baeyer-Villiger monooxygenases (BVMOs) have been shown to be powerful biocatalysts for synthetic organic chemistry applications and play key roles in the biosynthesis of various natural products. MtmOIV, a homodimeric FAD- and NADPH dependent BVMO, catalyzes a key frame-modifying step in the biosynthesis of the aureolic acid anticancer drug mithramycin (MTM). MTM recently gained renewed attention as an anticancer agent, because it uniquely targets oncogenic transcription factors as well as xenobiotic pumps of cancer stem cells. MtmOIV reacts with the highly complex intermediate premithramycin B via Baeyer-Villiger oxidation, which consequently leads to a C-C-bond cleavage that changes a biologically inactive molecular framework into an active one. Here we report the significantly improved protein crystal structure of MtmOIV, by itself and in complex with its natural substrate premithramycin B, which led to the identification of key residues important for substrate recognition and catalysis. Kinetic analyses of MtmOIV variants prepared by site-directed mutagenesis probed the substrate-binding pocket of MtmOIV and allowed us to locate the putative NADPH binding site. The work provides one of very few BVMO structures with a bound substrate, the first one of its unique fold and the first bound with its true natural substrate, and consequently paves the way to broaden the substrate specificity of MtmOV, necessary for the generation of new aureolic acid type anticancer agents.