Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes

Abstract

Following the recent discovery that heme proteins can catalyze the cyclopropanation of styrenyl olefins with high efficiency and selectivity, interest in developing new enzymes for a variety of non-natural carbene transfer reactions has burgeoned. The fact that diazo compounds and other carbene precursors are known mechanism-based inhibitors of P450s, however, led us to investigate if they also interfere with this new enzyme function. We present evidence for two inactivation pathways that are operative during cytochrome P450-catalyzed cyclopropanation. Using a combination of UV-Vis, mass spectrometry, and proteomic analyses, we show that the heme cofactor and several nucleophilic side chains undergo covalent modification by ethyl diazoacetate (EDA). Substitution of two of the affected residues with less-nucleophilic amino acids led to a more than two-fold improvement in cyclopropanation performance (total TTN). Elucidating the inactivation pathways of heme protein-based carbene transfer catalysts should aid in the optimization of this new biocatalytic function.

Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

“…In vitro, P411-VAC cis catalyzes N-vinylphthalimide cyclopropanation with a TOF of 22 min and 31% yield, 620 TTN, 91:9 dr and 93% ee, demonstrating significant decreases in all parameters, compared to whole-cell conditions (see Figure 2A, as well as Table S4 in the Supporting Information). This agrees with previous findings of enhanced hemoprotein carbene transfer activity in whole cells, compared to purified protein, 31,44,46 which we speculate is due to reduced EDA-induced protein alkylation and inactivation in the intact cells. Catalyst activity and lifetime is thus strongly influenced by the reaction (cellular) environment, as well as the protein sequence.…”

“…This long catalyst lifetime is notable among hemoprotein carbene transferases, as many of those described to date suffer from rapid inactivation through transfer of the reactive carbene intermediate to protein side chains or the porphyrin co-factor. 44,45 Indeed, such "burnout" behavior was observed for the evolved trans-selective variant P411-VAC trans , which shows much lower TTNs than P411-VAC cis and loses its catalytic activity after 30−60 min ( Figure 2B). Thus, P411-VAC cis may possess an active site structure that effectively directs iron carbenoid reactivity toward the intended alkene substrate while suppressing self-alkylation.…”

“…Up to 40 000 TTN were obtained at higher substrate or lower catalyst loadings (see Figure S4 in the Supporting Information), which places P411-VAC cis among the most active carbene transfer hemoproteins described to date. 31,33,44 For the trans-selective variant P411 BM3 -CIS I263G L437F, further site-saturation mutagenesis and screening yielded variant P411 BM3 -CIS I263G L437F V87L L181R, which we term P411-VAC trans . In whole E. coli cells, this variant catalyzes cyclopropanation of N-vinylphthalimide with EDA in 86% yield with 980 TTN, 3:97 dr, and 92% ee ( Figure 1B).…”

Hacking nature: genetic tools for reprograming enzymes

“…Impressively, it has also been possible to engineer wholly new catalytic functions for enzymes, such as direct amination of unactivated carbon atoms 14 , cyclopropanation 15 and Diels-Alder cycloaddition 16 (albeit, naturally-occurring enzymes for the latter reaction have since been discovered 17 ). Several stratagems have been used to introduce 'unnatural' functionality into enzymes, with examples of mechanism-based re-engineering of extant natural enzymes 6,14,15 and computer-aided de novo design of new active sites [18][19][20] .…”

“…Several stratagems have been used to introduce 'unnatural' functionality into enzymes, with examples of mechanism-based re-engineering of extant natural enzymes 6,14,15 and computer-aided de novo design of new active sites [18][19][20] . Often, enzymes constructed using the current iteration of such techniques have limited catalytic functionality; however, the catalytic properties of such synthetic and semisynthetic enzymes can be improved by direction evolution and related methodologies 21,22 .…”