Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization

Abstract

Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the tradeoff that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site-and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site-and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through twopoint binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site-and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.

Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products

“…In contrast, these conserved residues switch positions in biocatalysts that afford products with the S -configuration. We previously demonstrated that a two-point coordination of the phenolate substrate by Tyr237 and an arginine residue is critical for positioning the substrate within the active site in the R -selective enzyme, TropB. , To probe the role of Tyr118 in the stereoselectivity provided by AfoD, the AfoD Y118F variant was generated. AfoD Y118F was reacted with resorcinol S9 to afford dearomatized product in 53:47 ( S : R ) er (Figure S16).…”

State-of-the-Art Biocatalysis

“…Additional studies suggested two key active site residues are crucial in controlling the stereochemical outcome of the dearomatization reaction known to these proteins. 36 , 60 Though this does highlight a potential drawback of using SSNs in this way, the tool did ultimately demonstrate its utility in identifying other catalytically active proteins with desired activity. Work is currently underway to further characterize these enzymes in hopes of expanding our library of biocatalysts.…”

Tuning of pK values activates substrates in flavin-dependent aromatic hydroxylases

“…Our 19 F NMR studies demonstrate that the pK a of substrate is depressed in both a single-component and a two-component flavoprotein hydroxylase, supporting deprotonation of the phenol group as a general mechanism of substrate activation for electrophilic attack among these enzymes. Several one-component oxygenases were known to bind substrate in phenolate form (30,31,33,34), but we now place this on quantitative footing, demonstrating 9.1 and 14 kJ/mol stabilization of substrate phenolate consistent with electrostatic or hydrogen bonding mechanisms.…”