Regio- and stereoselective intermolecular oxidative phenol coupling of microbial P450 enzymes

M. MÜLLER; L. S. MAZZAFERRO; A. PRÄG; K. G. HUGENTOBLER; M. WILDE

Conferencia; VAAM Annual Meeting 2017; 2017

Cytochrome P450 enzymes (P450s) catalyze a broad variety of reactions, aside from hydroxylation reactions. The P450-catalyzed C?C bond-formation in oxidative phenol coupling often exhibits high regio- and/or stereoselectivity.In this work, the biosynthetic dimerization steps towards biaryl polyketides were investigated in bacterial and fungal strains. The PKS gene clusters for the biosynthesis of julichromes in spectomycin in S.spectabilis, and dimeric coumarins in Aspergillus spp. and Emericella desertorum were identified by genome analysis (Präg et al., 2014; Mazzaferro et al., 2015). Disruption of the PKS genes (julA-C) in S. afghaniensis led to complete loss of julichrome production. The gene julI encoding a cytochrome P450 enzyme was heterologous expressed in E. coli. The conversion of the monomer julichrome Q6 to the dimer julichrome Q6-6 verified that the P450 enzyme JulI is actually accountable for this coupling reaction. Likewise, the P450 enzymes KtnC from A. niger and DesC from E. desertorum were shown to catalyze the conversion of the monomer 7- demethylsiderin to the dimers orlandin and desertorin A, respectively, when expressed in S. cerevisiae.In consideration of a substrate reliance of the coupling reaction, variations of the natural substrate(s) were investigated and a core structure was deduced to generate a synthetic substrate. These experiments together with the on-going crystallization of JulI (in cooperation with the group of O. Einsle, Department of Biochemistry, Albert-Ludwigs-University, Freiburg) will bring insights into the substrate specificity and reaction selectivity of the P450-catalyzed phenol coupling.