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<p>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.
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Copper-catalyzed CuAAC/intramolecular C–H arylation sequence: Synthesis of annulated 1,2,3-triazoles