Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reaction

Abstract

The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in nature.

Figure 1. A designed enzyme for the Morita-Baylis-Hillman (MBH) reaction and the development of a dual function mechanistic inhibitor.

A) Chemical scheme of the MBH reaction between 1 and 2 catalysed by the computationally designed enzyme BH32. Intended design features include a His23 nucleophile positioned by a hydrogen bond acceptor, and hydrogen bond donors for oxyanion stabilization. B) Chemical scheme of BH32 inhibition with the mechanistic inhibitor 4. Addition of the His23 nucleophile is followed by E1cB elimination of the acetoxy group, generating a conjugated π-system, which can be monitored by an increase in absorbance at 325 nm. The stereochemistry of the exocyclic double bond in the inhibited complex is unknown. The wavelength scan shows the spectral changes that occur when BH32 (25 μM) is incubated with inhibitor 4 (250 μM) for 40 minutes.

Figure 2. Characterization of BH32, BH32.14 and selected variants.

A) Structure showing the amino acid positions mutated in BH32.14 (represented as spheres) mapped onto the structure of haloacid dehalogenase from Pyrococcus horikoshii (PDB code: 1X42). Mutations introduced during rounds 1-8 are shown in purple, rounds 9-12 in orange and rounds 13-14 in red. Position Asn14 was mutated twice (round 3 & 4) and is shown in purple, position Pro128 was mutated twice (round 5 & 14) and is shown in red. Computationally designed residues remaining following evolution are shown in blue. The His23 catalytic nucleophile is shown in stick representation. B) Bar chart showing the mean relative conversion (solid colour) and enantiomeric excess (hatched) achieved by selected variants along the evolutionary trajectory. Biotransformations were performed using 1 (3 mM), 2 (0.6 mM) and enzyme (18 μM), and analysed following 4.5 hours incubation (see Supplementary Table 2 for conversion and selectivity data). Error bars represent the standard deviation of measurements made in triplicate. C) Time course for the inhibition of BH32.8 (25 μM, purple), BH32 (25 μM, blue) and BH32 H23A (25 μM, black) with inhibitor 4 (250 μM). The black arrow indicates the time of protein addition. D) Bar chart comparing the turnover number (k _cat) of BH32 (blue), BH32.8 (purple), BH32.12 (orange) and BH32.14 (red) for the MBH reaction between 1 and 2. Steady state kinetic data (average of measurements made in triplicate at each substrate concentration) were fitted globally using a kinetic model for two substrates with randomly ordered binding to extract kinetic constants and associated errors (shown as error bars). Representative Michaelis-Menten plots at fixed concentrations of 1 or 2 are also shown in Supplementary Figure 2.

Figure 3. Substrate scope of engineered MBHases.

BH32.14 tolerates a range of activated alkenes, aldehydes and dialdehydes as substrates, leading to the production of densely functionalized MBH adducts with high conversions and selectivities. The less highly evolved BH32.8 accepts a broader range of substituents at the 2- and 3- positions, albeit with reduced efficiency and minimal enantioselectivity. Reported conversions and selectivities are the mean of biotransformations performed in triplicate. The stereochemistry of 5a-i, k-o & 5s-w are assigned by analogy to the (R)-3 product formed in BH32.14 mediated biotransformations. Biotransformations were performed using aldehyde/ketone (10 mM), activated alkene (50 mM for 3, 5a-c, 5f-h & 5j-w, 100 mM for 5d-e & 5i) and catalyst (0.5-5 mol%). Specific reaction conditions for the synthesis of 3 & 5a-w are presented in Supplementary Table 5.

Figure 4. Crystal structures of BH32 and BH32.12.

A ribbon representation of the superimposed coordinates of the BH32 design model (blue) and the evolved variant BH32.12 (orange). The His23 nucleophile and catalytic Arg124 from BH32.12 are shown in orange stick representation. Substrates docked into BH32.12 are shown in all atom coloured stick representation. The active site surface volume of the BH32 design model is shown as a green transparency whilst the equivalent active site surface volume for BH32.12 is shown as a grey mesh. The right hand panels show a close-up representation of the active sites. The top panel shows the substrate binding pocket of BH32 design model highlighting the spatial arrangement of the designed residues Gln128, Ser124 and His23. The protein backbone is shown in ribbon representation (blue) with the protein surface shown in grey. The substrates are derived from the original composite transition state model with the aldehyde shown in stick representation and transparent yellow CPK spheres. 2-Cyclohexen-1-one is also shown in stick representation with accompanying transparent green CPK spheres. The bottom panel shows the aldehyde and enone binding pocket of BH32.12 highlighting the spatial arrangement of key residues Trp88, Trp10, Arg124 and His23 (stick representation - orange carbon atoms). The protein backbone is shown in ribbon representation (orange) with the protein surface shown in grey. The substrate positions depicted are those obtained from initial docking studies prior to DFT calculations. The aldehyde substrate is shown in stick representation with accompanying transparent pink CPK spheres. 2-Cyclohexen-1-one is shown in stick representation with transparent magenta CPK spheres.

Figure 5. Proposed catalytic mechanism of an engineered MBHase.

A) DFT states for intermediates 1, 2 & 3 are shown in all atom coloured stick representation. The images presented show only a subset of the atoms used in the full DFT calculation for greater visual clarity. Atoms derived from several residues that form close packing interactions with substrates 1 and 2 during catalysis (Trp10, Trp88, Arg124 & Ser129) are highlighted with grey dot surfaces. Heavy atoms from the substrates are highlighted with magenta transparent CPK spheres. Atoms from the His23 nucleophile and catalytic Arg124 are shown in all atom coloured stick representation. The hydrogen bonding network that emerged during evolution between Trp10, Ser129 and the aldehyde substrate are shown as black dashed lines. In addition, hydrogen bonds between Arg124 and each intermediate state are shown. B) The BH32.14 catalytic mechanism showing the role played by Arg124 in stabilizing three intermediates covalently bound through His23. A catalytic water molecule is shown in blue to facilitate proton transfer from C2 to the C3 alkoxide.