Mechanistic Analysis of Stereodivergent Nitroalkane Cyclopropanation Catalyzed by Nonheme Iron Enzymes

Abstract

BelL and HrmJ are α-ketoglutarate-dependent nonheme iron enzymes that catalyze the oxidative cyclization of 6-nitronorleucine, resulting in the formation of two diastereomeric 3-(2-nitrocyclopropyl)alanine (Ncpa) products containing trans-cyclopropane rings with (1'R,2'R) and (1'S,2'S) configurations, respectively. Herein, we investigate the catalytic mechanism and stereodivergency of the cyclopropanases. The results suggest that the nitroalkane moiety of the substrate is first deprotonated to produce the nitronate form. Spectroscopic analyses and biochemical assays with substrates and analogues indicate that an iron(IV)-oxo species abstracts proS-H from C4 to initiate intramolecular C-C bond formation. A hydroxylation intermediate is unlikely to be involved in the cyclopropanation reaction. Additionally, a genome mining approach is employed to discover new homologues that perform the cyclopropanation of 6-nitronorleucine to generate cis-configured Ncpa products with (1'R,2'S) or (1'S,2'R) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.

Figure 1.

(a) Distribution of 3 and 4 detected by ¹³C-NMR using [6-¹³C]-3 as the substrate and (b) SscBelL catalyzed reaction efficiency at various pH conditions.

Figure 2.

Spectroscopic evidence of C4-H activation by a ferryl intermediate during SscBelL catalyzed cyclopropanation. Top: selected spectra demonstrating the optical feature changes upon rapid mixing of the SscBelL•Fe(II)•αKG•3 with O₂ saturated buffer. The decay and reformation of the optical feature centered at ~520 nm is accompanied by the formation and decay of a broad UV feature (ca. 300–400 nm). The inset shows the absorption change at 330 nm with 3 or [4,4-²H₂]-3. Bottom: Mössbauer spectra of samples generated by freeze-quench technique on the selected time points from the reaction with 3 (left) or with or [4,4-²H₂]-3 (right). The grey solid lines represent the overall spectral simulations. The red solid lines represent the spectral component of the ferryl intermediate with isomer shift (δ) of 0.27 mm/s and quadrupole splitting (|ΔE_Q|) of 0.80 mm/s. See Table S2 and S3 for detailed simulation parameters.

Figure 3.

(a) LCMS analysis of the cyclopropanation reactions catalyzed by BelL homologues. EIC traces corresponding to [M + H]⁺ signals from 1-Dns (m/z = 408.1) are shown. (b) ¹³C NMR spectra of HrmJ, BelL, SscBelL and SrBelL catalyzed reactions using [6-¹³C]-3 as substrate.

Figure 4.

Comparison of residues around the active sites of the tested homologues. Indicated residue numbers are those of BelL. Stereochemistry of the main product(s) are also shown.

Figure 5.

LCMS analysis of the cyclopropanation reactions catalyzed by BelL and HrmJ variants EIC traces corresponding to [M + H]⁺ signals from 1-Dns (m/z = 408.1) are shown in red.

Figure 6.

HPLC analysis of the reactions catalyzed by BelL variants. The reaction mixture was treated with dansyl chloride prior to the analysis. UV 340 nm chromatogram were shown.

Scheme 1.

Stereodivergent cyclopropanation reactions catalyzed by BelL and HrmJ during biosynthesis of hormaomycin and belactosin A.

Scheme 2.

Possible mechanisms for the BelL and HrmJ-catalyzed cyclopropanation of 6-nitronorleucine (3)