Epoxidation and Late-Stage C–H Functionalization by P450 TamI Are Mediated by Variant Heme-Iron Oxidizing Species

Abstract

P450-catalyzed hydroxylation reactions are well understood mechanistically including the identity of the active oxidizing species. However, the catalytically active heme-iron species in P450 iterative oxidation cascades that involve mechanistically divergent pathways and distinct carbon atoms within a common substrate remains unexplored. Recently, we reported the enzymatic synthesis of trifunctionalized tirandamycin O (9) and O′ (10) using a bacterial P450 TamI variant and developed mechanistic hypotheses to explore their formation. Here, we report the ability of bacterial P450 TamI L295A to shift between different oxidizing species as it catalyzes the sequential epoxidation, hydroxylation, and radical-catalyzed epoxide-opening cascade to create new tirandamycin antibiotics. We also provide evidence that the TamI peroxo-iron species could be a viable catalyst to enable nucleophilic epoxide opening in the absence of iron-oxo compound I. Using site-directed mutagenesis, kinetic solvent isotope effects, artificial oxygen surrogates, end-point assays, and density functional theory (DFT) calculations, we provide new insights into the active oxidant species that P450 TamI employs to introduce its unique pattern of oxidative decorations.

Enzymatic Control over Reactive Intermediates Enables Direct Oxidation of Alkenes to Carbonyls by a P450 Iron-Oxo Species

“…Established catalysts for alkene epoxidation via high-valent metal-oxo complexes include Jacobsen’s manganese­(III)­salen complexes, synthetic metalloporphyrins, biomimetic non-heme iron complexes, − and enzymes such as heme-dependent cytochrome P450 monooxygenases and peroxygenases or non-heme α-ketoglutarate-dependent dioxygenases . The exact mechanism of metal-oxo-mediated alkene epoxidation has long been controversial. ,,,− A currently widely accepted view is that epoxide formation proceeds via an almost concerted reaction mechanism, potentially through an extremely short lived radical intermediate (Scheme ). Support for a concerted nature of the oxo transfer or a two-step mechanism in which the second step occurs very fast comes from the retention of the stereochemistry in epoxidation of cis -alkenes. ,,, It is believed that metal-oxo-mediated alkene to carbonyl oxidation proceeds from the radical intermediate via an electron/hydride transfer process including the formation of a carbocation species (Scheme ).…”

Efficient Production of Epoxy‐Norbornane from Norbornene by an Engineered P450 Peroxygenase

“…This study involves a new substrate for epoxidation catalyzed by P450 enzymes. P450 enzymes catalyze epoxidation reactions of chain terminal alkenes, [11,30] styrene, [31] and terpene derivatives, [32] and are used in the practical production of various high‐value compounds, such as tirandamycin [33] and mycinamicin [34] . Using NBE as a substrate, this study developed an efficient biological protocol for producing EPO‐NBE.…”

Asymmetric Bio‐epoxidation of Unactivated Alkenes

“…The Sherman group reported that TamI from Streptomyces sp. 307‐9 catalyzed the iterative oxidation of three positions of the bicyclic ketal moiety of tilanamycin C 44 , namely C10 hydroxylation, C11/C12 epoxidation, and C18 hydroxylation [70–73] . It is shown that TamI has good regio‐ and stereoselectivity for the oxidation of tilanomycin C 44 , and at the same time emphasizes the important role of multifunctional natural enzymes in catalyzing the synthesis of natural products.…”