Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C–H Functionalization and Epoxidation of Tirandamycin Antibiotics

Abstract

Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent new tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244 and/or Leu295, that alter the native selectivity, step sequence and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity enabling catalyst-controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five new, bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights on the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI. File list (2) download file view on ChemRxiv TamI_mutants_final.pdf (3.51 MiB) download file view on ChemRxiv SI_TamI_mutants_final.pdf (12.90 MiB) Engineering P450 TamI as an iterative biocatalyst for selective late-stage C-H functionalization and epoxidation of tirandamycin antibiotics

Bacterial Associates of a Desert Specialist Fungus-Growing Ant Antagonize Competitors with a Nocamycin Analog

“…The complex oxidation patterns observed for the polycyclic ketal headgroups of the nocamycins and tirandamycins have inspired mechanistic studies to untangle the sequence of oxidative tailoring steps. 19,[26][27][28][29][30] Remarkably, a single cytochrome P450 enzyme, TamI, accomplishes three distinct oxidative steps as part of a cascade of oxidative reactions on the tirandamycin scaffold, resulting in installation of a hydroxyl group at C18, analogous to the C20 hydroxyl of nocamycin V. 28,29 From a phylogeny of cytochromes P450 from tirandamycin and nocamycin BGCs (Figure S5), we find that NmvR is homologous to TamI, which could indicate a role in installation of the C20 hydroxyl group. A complex interplay of oxidations is likely and future investigation of the biosynthesis of nocamycin V would contribute to our understanding of multifunctional enzymes and co-dependent oxidations.…”

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

“…Continuing their work with the iterative cytochrome P450, TamI, Sherman et al engineered mutant L244A_L295V capable of catalysing an unprecedent four-step oxidative biocatalytic cascade on tirandamycin C to generate biologicallyactive tirandamycin B, overriding the enzyme's innate need for its oxidative partner, TamL. [173] Recently, Sewald et al have shown that FMOs can be successfully integrated in biocatalytic cascades to form an array of indigoids. [206] Starting with L -tryptophan, the group employed crosslinked enzyme aggregates for C-5, C-6 or C-7 bromination of the indole moiety, followed by cleavage of the amino acid backbone catalysed by tryptophanase (TnaA), to give bromoindole.…”

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

“…Cytochrome P450 enzymes (P450s) are a superfamily of heme proteins involved in drug metabolism, xenobiotic detoxification, and steroid biosynthesis [ 1 , 2 ]. P450s are promising biocatalysts for organic synthesis, drug discovery, and bioremediation because of their versatile catalytic oxyfunctionalizations of a variety of substrates [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. However, one of the limitations of applying P450s as in vitro biocatalysts is that most P450s require reduced nicotinamide cofactors NAD(P)H and a complex system of electron transport partners (redox partners) to activate molecular oxygen for the formation of active species.…”