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

Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent useful 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 bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.

Molecular Basis of Iterative C─H Oxidation by TamI, a Multifunctional P450 monooxygenase from the Tirandamycin Biosynthetic Pathway.

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C─H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C─H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C─H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.