Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

Abstract 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 activation and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven to be 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 hydroxylation 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 (MD) simulations and targeted mutagenesis, suggests that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. Quantum mechanics calculations and MD simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity and pattern of regio- and stereoselectivity in catalyzing the three-step oxidative cascade.