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 <i>trans</i>-cyclopropane rings with (1'<i>R,</i>2'<i>R</i>) and (1'<i>S,</i>2'<i>S</i>) 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 pro<i>S</i>-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 <i>cis</i>-configured Ncpa products with (1'<i>R,</i>2'<i>S</i>) or (1'<i>S,</i>2'<i>R</i>) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.