Page 1 of 5 Journal of the American Chemical Society 1 2 3 4 5 6 7 Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and 8 Enhanced by Non-Native Small-Molecule Ligands 9 10 Nathaniel W. Goldberg†,§, Anders M. Knight‡,§, Ruijie K. Zhang†,‖, Frances H. Arnold†,‡,* 11 12 †Division of Chemistry and Chemical Engineering and ‡Division of Biology and Bioengineering, California Institute of 13 Technology, 1200 East California Boulevard, MC 210-41, Pasadena, California 91125, United States 14 Supporting Information Placeholder 15 16 ABSTRACT: Transition-metal catalysis is a powerful tool for the 17 construction of chemical bonds. Here we show that Pseudomonas O O O O S L S Ph savastanoi ethylene-forming enzyme (PsEFE), a non-heme iron N3 N 18 Fe 19 enzyme, can catalyze olefin aziridination and nitrene C–H N N insertion, and that these activities can be improved by directed O O N O N 20 O O evolution. The non-heme iron center allows for facile modification S O N S 21 of the primary coordination sphere by addition of metal- 3 NH 22 coordinating molecules, enabling control over enzyme activity and 23 selectivity using small molecules. 24 O O O H O O 25 L = - - - N - - 26 O O O O O Over the last century, chemists have developed myriad synthetic O O 27 transition-metal catalysts to access new chemical transformations Figure 1. Small-molecule activation of a non-heme iron center for 28 and modes of reactivity. Nature has been developing catalysts for nitrene transfer. Carboxylate-containing ligands α-ketoglutarate, 29 far longer: over billions of years, she has evolved a rich repertoire N-oxalylglycine, and acetate modulate the nitrene-transfer activity 30 of proteins that perform most of the chemical reactions of life. But of variants of P. savastanoi ethylene-forming enzyme. nature’s inventions do not include many of the best inventions of 31 To search for abiological catalytic promiscuity among natural 32 human chemists. Our efforts to merge abiological transition-metal chemistry with nature’s vast toolbox of metalloproteins have metalloproteins, we looked at α-ketoglutarate (αKG)-dependent 33 focused on heme-binding proteins1, as the heme cofactor and its iron enzymes, a family of enzymes which features a conserved 34 metal-binding active site with iron coordinated by two histidines analogues are well-studied in synthetic transition-metal chemistry. 4 35 However, heme-binding proteins represent only a small fraction of and one aspartate or glutamate . In nature, these enzymes perform 36 the chemical diversity present in natural metalloproteins. similar chemistry to the heme-binding cytochrome P450 family, in which a high-valent iron-oxo intermediate performs C–H 37 Metalloproteins comprise greater than 30% of all proteins2 and are responsible for some of the most fundamental chemical reactions hydroxylation, olefin epoxidation, or other oxidative 38 5 in biology, including nitrogen fixation, photosynthesis, and DNA transformations . Though members of this enzyme family have 39 synthesis. Natural metalloproteins bind a variety of metals in a wide been reported to catalyze reactions beyond their native functions, 40 all the reactions reported proceed through the native iron-oxo range of metal-binding sites, either coordinating the metal ion itself 6 41 or a more complex metal-containing cofactor. Nearly any mechanism . We hypothesized that non-heme iron enzymes might 42 heteroatom-containing side chain can coordinate to a metal, in also be able to catalyze abiological transformations similar to heme-binding proteins through a non-natural mechanistic pathway. 43 addition to the peptide backbone, allowing myriad possible 3 44 coordination environments . Many coordination environments in We screened a set of seven purified α-ketoglutarate (αKG)- dependent iron dioxygenases against the intermolecular 45 non-heme metalloenzymes have multiple open coordination sites at the metal center, a key feature of numerous synthetic transition- aziridination reaction of styrene 1 and p-toluenesulfonyl azide 2. 46 metal catalysts. Expanding new-to-nature catalysis to non-heme Aziridination7 and carbon–hydrogen (C–H) bond insertions of 47 metalloenzymes would open a new world of transition-metal nitrenes8 have been reported using engineered heme-binding 48 biocatalysis. In this work we show that a non-heme iron enzyme proteins and were subsequently proposed in a natural product 49 can catalyze nitrene-transfer chemistry (Figure 1). This non-native biosynthetic pathway9. Chang and coworkers speculated the 50 activity is enhanced by binding of non-native small-molecule existence of a transient iron-nitrene intermediate in their report of ligands and has been improved by directed evolution. the transformation of alkyl azides to nitriles by an αKG-dependent 51 iron dioxygenase, but this reaction still proceeds through the 52 canonical iron-oxo catalytic cycle6b. To date, no reported non-heme 53 iron enzyme, natural or engineered, has been reported to catalyze 54 productive nitrene transfer. 55 From the set of enzymes we tested, only Pseudomonas 56 savastanoi ethylene-forming enzyme (PsEFE, UniProt ID 57 P32021), formed aziridine 3 significantly above background 58 59 60 ACS Paragon Plus Environment Journal of the American Chemical Society Page 2 of 5 (Supplementary Table S2). Compared to other members of the Table 1. Aziridination catalyzed by wild-type PsEFE 1 αKG-dependent iron dioxygenase family PsEFE is mechanistically 2 and structurally distinct. While most enzymes of this family O O catalyze the oxidation of a substrate, often C–H hydroxylation, S N 3 N S PsEFE natively catalyzes the fragmentation of the usual co- + 3 4 O O substrate α‑ketoglutarate to ethylene, as well as the hydroxylation 5 10 of L-arginine . Structurally, PsEFE possesses a hybrid fold, 1 2 3 6 combining elements of both type I and type II αKG-dependent iron 1 7 enzymes. It binds α-ketoglutarate in a strained conformation in an Deviation from standard conditions Relative activity 8 unusually hydrophobic pocket, which is likely responsible for the None 1.00 11 9 atypical catalytic activity . No iron 0.08 As the iron-binding site in PsEFE is quite unlike that of the 10 No αKG 0.52 11 heme-binding proteins that perform nitrene-transfer chemistry, we Acetate2 instead of αKG 6.72 12 sought to characterize the necessary components of the reaction. Iron is required, with a single added equivalent of iron(II) sufficient N-oxalylglycine3 instead of αKG 7.75 13 to fully restore catalytic activity of the wild-type apoenzyme. 14 PsEFE has three coordination sites filled by amino-acid side chains Acetate instead of αKG, no ascorbic acid 6.01 15 (two histidines and one aspartate), leaving up to three additional 1Standard conditions: Reactions were performed in MOPS 16 sites open for binding. In the native catalytic mechanism of PsEFE buffer (20 mM pH 7.0) with 5% ethanol co-solvent, with 20 μM 17 and other members of its family, α-ketoglutarate occupies two of apoenzyme, 1 mM Fe(NH ) (SO ) , 1 mM αKG (as disodium these sites and is required for activity, as it is oxidatively 4 2 4 2 18 salt), 1 mM L-ascorbic acid, and 10 mM 1 and 2. 2Sodium salt. decarboxylated to succinate to generate the reactive iron-oxo 3Free acid. 19 intermediate. PsEFE has been shown to catalyze arginine 20 hydroxylation with α-ketoadipate instead of α-ketoglutarate, but We then sought to improve PsEFE for aziridination via directed 21 with 500-fold lower activity. Other α-ketoacids were reported to evolution, targeting active-site residues with site-saturation 22 give no activity12. Nitrene transfer, however, does not proceed mutagenesis and screening for enhanced activity. Details of our 23 through the native catalytic cycle and therefore does not require engineering strategy are in the Supporting Information. Initial 24 αKG as a co-substrate; the αKG is now more a ligand and as such screening for the first round was performed in the presence of could potentially be replaced by different small-molecule ligands. exogenous α-ketoglutarate, but for validation of this round and for 25 Intrigued by the possibility of modulating enzyme activity by all subsequent evolution we screened with exogenous acetate. 26 changing the primary coordination sphere of the catalytic iron, we Acetate was chosen as the preferred ligand because it enhanced the 27 tested PsEFE for aziridination with a set of α-ketoglutarate mimics activity of the wild-type enzyme significantly more than the native 28 and related molecules as additives. We found that whereas addition α-ketoglutarate, it is biologically ubiquitous, and it is inexpensive. 29 of a carboxylate is beneficial for activity (though not required), the Although α-ketoglutarate is the native ligand and is naturally 30 wild-type enzyme is significantly more active for aziridination with present at near-millimolar intracellular concentration in added acetate or N-oxalylglycine (NOG, a general αKG-dependent Escherichia coli13, we reasoned that by supplementing the reaction 31 enzyme competitive inhibitor4) compared to α-ketoglutarate (Table medium with acetate we could evolve PsEFE to be dependent on 32 1). acetate instead, despite screening under whole cell or lysate 33 conditions. 34 After two rounds of site-saturation mutagenesis and one round 35 of recombination, we found a variant with five mutations from the 36 wild type (T97M R171L R277H F314M C317M, PsEFE 37 MLHMM) which catalyzed the formation of 3 with 120 total 38 turnover number (TTN) and 88% enantiomeric excess (ee) favoring the (R)-enantiomer (Figure 2A). Four of the five introduced 39 mutations are in the binding pocket of the native substrate arginine 40 and presumably are involved in substrate binding. The fifth 41 beneficial mutation is at Arg-277, the residue whose guanidino 42 group natively binds the distal carboxylate of α-ketoglutarate 43 (Figure 2B).