Enzymatic hydroxylation of an unactivated methylene C-H bond guided by molecular dynamics simulations

Abstract

The hallmark of enzymes from secondary metabolic pathways is the pairing of powerful reactivity with exquisite site selectivity. The application of these biocatalytic tools in organic synthesis, however, remains under-utilized due to limitations in substrate scope and scalability. Here, we report how the reactivity of a monooxygenase (PikC) from the pikromycin pathway is modified through computationally guided protein and substrate engineering, and applied to the oxidation of unactivated methylene C-H bonds. Molecular dynamics and quantum mechanical calculations were used to develop a predictive model for substrate scope, site selectivity and stereoselectivity of PikC-mediated C-H oxidation. A suite of menthol derivatives was screened computationally and evaluated through in vitro reactions, where each substrate adhered to the predicted models for selectivity and conversion to product. This platform was also expanded beyond menthol-based substrates to the selective hydroxylation of a variety of substrate cores ranging from cyclic to fused bicyclic and bridged bicyclic compounds.

Figure 1

Selected oxidation products of (−)-menthol and calculated bond dissociation energies (BDEs) for the corresponding C–H bonds in kcal mol⁻¹. The calculated BDEs display a general trend in C–H bond strength, tertiary < secondary < primary.

Figure 2

Substrate anchoring mechanism employed by P450 PikC. (a) Co-crystal structure of natural substrate, YC-17, and PikC (PDB ID 2CD8) depicting the anchoring salt bridge between E94 and the dimethylamino group of YC-17. (b) Evolution of the PikC substrate-engineering approach with the natural anchoring group, desosamine and dimethylamine containing synthetic anchoring groups.

Figure 2

Substrate anchoring mechanism employed by P450 PikC. (a) Co-crystal structure of natural substrate, YC-17, and PikC (PDB ID 2CD8) depicting the anchoring salt bridge between E94 and the dimethylamino group of YC-17. (b) Evolution of the PikC substrate-engineering approach with the natural anchoring group, desosamine and dimethylamine containing synthetic anchoring groups.

Figure 3

The persistence of the potential salt bridges (>4 Å) between a given substrate anchoring group and different PikC residues (percentages measured from MD simulations) are shown; (+)/(−) distinction refers to menthol absolute configuration.

Figure 4. Substrate binding and tertiary structure of PikC variants observed in MD simulations

(a) Improper binding of substrate (−)-21 to PikC_wt showing detrimental interactions with E246, absence of contacts with E94, E85 or E48, and disruption of the hydrogen bond network in the R172/E146/W150 motif. (b and c) PikC_wt in open conformation showing an open active site with widely spread binding residues (E94, E85 and E48). (d) Adequate binding of substrate (−)-26 to PikC_{D50ND176QE246A} showing interactions with the all three binding residues simultaneously and the undistorted R172/E146/W150 motif. (e and f) PikC_{D50ND176QE246A} in closed conformation showing a narrow active site channel and a tight arrangement of the binding residues.

Figure 5. Stereoselectivity in the C4–H abstraction catalyzed by PikC

(a) Quantum mechanically (QM) optimized transition for hydrogen abstraction of H4_eq in (−)-menthol (see Supplementary Fig. 2). (b) Representative snapshot of a MD trajectory of PikC_D50N bound to 25 showing the substrate in close proximity to the catalytic heme iron-oxo species (Compound I). (c) and (d) Orientation of H4_eq (in green) and H4_ax (in orange) of (−)-menthol (c) and (+)-menthol (d) relative to the iron-heme oxo group in PikC_D50N, derived from 0.5 µs MD trajectories. Each point represents a simulation snapshot. Deviations of the distances (x axis) and angles (y axis) from the quantum mechanically optimized transition structure (in blue) with a hypothetical model heme catalyst are shown. The greater separation between the two sets of points (H4_eq and H4_ax) in (−)-menthol is interpreted as a potential higher diastereoselectiviy in the associated transition states, and thus the hydroxylation reaction.

Figure 6

Site selective oxidation with PikC_{D50ND176QE246A}-RhFRED. C–O bonds highlighted in red = hydroxyl group introduced in the P450 reaction. Numbers given in parentheses are TTN values determined in triplicate. Reaction conditions: 1 mM substrate, 5 µM enzyme, 1 mM NADP⁺, 5 mM glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase.