Journal
ACS CATALYSIS
Volume 11, Issue 13, Pages 8304-8316Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.1c01460
Keywords
biocatalysis; enzyme engineering; cytochrome P450; C-H functionalization; natural products; antibiotics
Categories
Funding
- National Science Foundation under the CCI Center for Selective C-H Functionalization [CHE-1700982]
- National Institutes of Health [R35 GM118101, R35 GM118133]
- Hans W. Vahlteich Professorship
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Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. The study reports the structure of the multifunctional bacterial P450 TamI and the design of a toolbox of TamI biocatalysts, including a variant capable of catalyzing a four-step oxidative cascade without assistance. These tuned enzymes enable catalyst-controlled C-H functionalization and alkene epoxidation, leading to the synthesis of bioactive tirandamycin derivatives. Quantum mechanics calculations and MD simulations provide insights into the altered selectivity and enhanced oxidation mechanisms of the iterative P450 TamI.
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.
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