A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase

Abstract

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

Keywords: biocatalysis; biocatalytic ring expansion; carbene transfer; de novo protein design; enzyme design.

Fig. 1.

Metallocarbenoid formation and its reactive potential within a de novo-designed c-type cytochrome maquette, C45. (A) Single snapshot from a 1-μs molecular-dynamics simulation of the C45 maquette (6). (B) Formation and potential reactivity of a heme-based metallocarbenoid intermediate, illustrating aldehyde olefination, olefin cyclopropanation, and amine N–H insertion reactions. (C) UV/visible EDA-treated C45 (red), engineered Rma-TDE (green), and MP-11 (blue) obtained by rapid mixing experiments in a stopped-flow spectrophotometer. The putative metallocarbenoid species was generated by mixing 2.5 mM EDA with 7.5 μM ferrous heme protein in 100 mM KCl and 20 mM CHES, pH 8.6 (10% EtOH).

Fig. 2.

Stability and reactivity of the metallocarbenoid:C45 intermediate. (A) Time course of electronic spectra recorded following rapid mixing of ferrous C45 (red) with EDA in 10% EtOH at 5 °C. The appearance of the metallocarbenoid intermediate (blue) is concomitant with the disappearance of the ferrous C45 spectrum. Spectra presented were recorded 1, 2, 4, 6, 8, 10, 15, 20, 50, and 100 s after mixing. (B) Metallocarbenoid formation and stability in the absence of styrene substrate. Single-wavelength traces represent the time course of ferrous C45 (418 nm; blue; 7.5 μM protein, 10% EtOH) and metallocarbenoid:C45 adduct (437 nm; red) following rapid mixing of ferrous C45 with 500 μM EDA at 5 °C. Once formed, the metallocarbenoid:C45 adduct persists for the duration of the experiment (1,000 s). (C) Electronic spectra of metallocarbenoid intermediates formed between ferrous C45 and EDA, BnDA, and ^tBuDA following rapid mixing in the stopped-flow apparatus. (D) EDA-concentration-dependent formation of the C45 metallocarbenoid adduct. Kinetic data were recorded by using a stopped-flow spectrophotometer and analyzed as described in Experimental Details. The limiting rate constant (k_lim) and pseudo-Michaelis constant (K₁) for metallocarbenoid formation are 0.50 s⁻¹ and 74 μM, respectively. Data were collected in triplicate, and error bars represent the SD.

Fig. 3.

Reactivity of the metallocarbenoid intermediates of C45 and Rma-TDE. (A and B) Electronic spectra of C45 (A) and Rma-TDE (B) recorded after rapid mixing of ferrous heme protein (7.5 μM; red trace) with EDA (500 μM) and styrene (3 mM) at 5 °C in the stopped-flow spectrophotometer. Green traces correspond to spectra recorded at time points where the maximum quantity of metallocarbenoid was accumulated; blue traces correspond to the final spectra recorded in the experiments. (C and D) Metallocarbenoid formation and decay of C45 (C) and Rma-TDE (D) in the presence of styrene. Single-wavelength traces represent the time course of ferrous heme protein (blue traces) (7.5 μM protein) and metallocarbenoid adduct (red) following rapid mixing of ferrous heme protein with 500 μM EDA and 3 mM styrene at 5 °C. (E and F) Electronic difference spectra highlighting the spectroscopic changes associated with metallocarbenoid formation and decay for C45 (E) and Rma-TDE (F). The lower, red traces demonstrate the spectroscopic changes that occur during the formation of the metallocarbenoid, which are identical to those observed during its subsequent decay (upper, blue traces). These indicate reformation of the initial ferrous species after carbene insertion and after EDA is exhausted.

Fig. 4.

Carbene transferase activity of C45. (A) Cyclopropanation of substituted styrenes catalyzed by C45. TTNs and % ee for each combination of ferrous C45 with para-substituted styrenes and functionalized diazoacetates are shown. Only the (R,R) cyclopropanated product is displayed in the reaction scheme, representing the dominant product in all cases. All reactions were carried out with 0.1% catalyst loading (10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 10 mM diazo compound, and 30 mM substituted styrene in 100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH/H₂O. (B) N–H insertion of primary and secondary amines catalyzed by C45. Only the monofunctionalized product of the p-chloroaniline insertion reaction is shown, and the corresponding TTN is calculated based on the yield of both monosubstituted and disubstituted products. All reactions were carried out with 0.1% catalyst loading (10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 10 mM EDA, and 30 mM amine substrate in 100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH/H₂O. (C) Olefination of substituted benzaldehydes catalyzed by C45. All reactions were carried out with 0.1% catalyst loading (10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 10 mM PPh₃, 10 mM EDA, and 30 mM substituted benzaldehyde in 100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH/H₂O.

Fig. 5.

Heteroaromatic ring expansion catalyzed by C45. (A) Reaction scheme for the ring-expansion strategy using ethyl 2-bromo-2-diazoacetate, pyrrole, and ferrous C45. Following carbene transfer to the pyrrole, spontaneous rearrangement of the bicyclic ring system leads to elimination of HBr and formation of a 6-membered pyridine ring. (B) C18 reversed-phase HPLC traces of the C45-catalyzed ring expansion of pyrrole to ethyl nicotinate. Traces 1 and 2 show the C45-catalyzed ring expansion compared to a partially hydrolyzed commercial standard of ethyl nicotinate. The ring expansion was carried out with 1% catalyst loading (10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 1 mM ethyl 2-bromo-2-diazoacetate, and 10 mM pyrrole in 100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH. Traces 3 to 5 show the results of incubating whole cells containing the C45 expression vector and pEC86 harboring the E. coli cytochrome c maturation apparatus. Traces 3 and 4 represent reactions between whole cells, pyrrole, and ethyl 2-bromo-2-diazoacetate at 3 and 6 h after inoculation and in the absence of the inducer, IPTG. Trace 5 represents the reaction with C45-expressing whole cells, pyrrole, and ethyl 2-bromo-2-diazoacetate. In this case, the cells were grown for 3 h and induced with 1 mM IPTG, and C45 was expressed for a further 3 h prior to use in the whole-cell transformation. Reaction conditions are fully described in Experimental Details.