Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction

Abstract

The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.

Figure 1. The Diels-Alder reaction

Diene (1) and dienophile (2) undergo a pericyclic [4+2] cycloaddition (3) to form a chiral cyclohexene ring (4). Also shown in (3) is a schematic of the design target active site, with hydrogen bond acceptor and donor groups activating the diene and dienophile and a complementary binding pocket holding the two substrates in an orientation optimal for catalysis.

Figure 2. Structure of a Designed Diels-Alderase

Top panel (2A): surface view of the design model (DA_20_00, green) bound to the substrates (diene and dienophile, purple). The catalytic residues making the designed hydrogen bonds are depicted as sticks. Middle panel (2A): overlay of the design model (DA_20_00, green) and the apo enzyme crystal structure of DA_20_00_A74I (brown). Bottom panel (2C): Contribution of designed residues to catalysis assessed through reversions back to the native amino acid at each position individually. Colors indicate the reduction in activity upon reversion to the native amino acid; 2 fold (blue) to > 10 fold (red). The figure was generated with PyMol (15).

Figure 3. Kinetic Characterization

(A) Dependence of reaction velocity for DA_20_10 on diene concentration for different fixed dienophile concentrations. The diene concentration was varied from 3.0 to 0.18 mM with a fixed concentration of 100 mM (◢), 66 mM (■), 44 mM (◆), 30 mM(▲), 20 mM(▼), or 13 mM (●) dienophile (B) Dependence of reaction velocity for DA_42_04 at varying diene concentrations for different fixed dienophile concentrations. The diene concentration was varied from 2.0 to 0.06 mM with a fixed concentration of 100 mM (◢), 50 mM (■), 25 mM (◆), 13 mM(▲), 7 mM(▼), or 3 mM (●) dienophile. Reaction conditions are described in the supplementary material.

Figure 3. Kinetic Characterization

(A) Dependence of reaction velocity for DA_20_10 on diene concentration for different fixed dienophile concentrations. The diene concentration was varied from 3.0 to 0.18 mM with a fixed concentration of 100 mM (◢), 66 mM (■), 44 mM (◆), 30 mM(▲), 20 mM(▼), or 13 mM (●) dienophile (B) Dependence of reaction velocity for DA_42_04 at varying diene concentrations for different fixed dienophile concentrations. The diene concentration was varied from 2.0 to 0.06 mM with a fixed concentration of 100 mM (◢), 50 mM (■), 25 mM (◆), 13 mM(▲), 7 mM(▼), or 3 mM (●) dienophile. Reaction conditions are described in the supplementary material.

Figure 4. Absolute stereoselectivity of DA_20_10

The transition states which lead to the four possible ortho-stereoisomers are shown above the reaction chromatograms. Background reaction: 2 mM diene and 70 mM dieneophile in a PBS solution for 24 hours at 298 K. DA_20_10 reaction: 50 μM protein, 0.5 mM diene, and 10 mM dieneophile in a PBS solution for 48 hours at 298K (16).

Figure 5. Designed Control of Substrate Specificity

Reactions were carried out with 0.2 mM diene 1 (Figure 1), and 10 mM of one of the six dienophiles depicted above in PBS at 298K in the absence or presence of 60 μM DA_20_10 or DA_20_10_H287N. The values in the figure are the mean (colored bars) and standard deviation (error bars) of four independent measurements of the product peak area (arbitrary units) formed per hour (16).