4. Expanding new-to-nature reactions beyond heme proteins
4.4 Nitrene-transfer activity with αKG enzymes
research groups are focused on improving the expression and stability of these proteins in model organisms, however, and these more robust expression platforms could be tested as potential starting points. As more non-heme metalloproteins are engineered for other new- to-nature reactions on model substrates like those tested in this section, those variants can be screened for their activity on hydrosilylation and related reactions. Once initial activity is found, it has been shown countless times that directed evolution can improve that activity – all that is needed is a starting point and a good screen.36
Figure 4-7. Typical oxene-transfer reactions carried out by non-heme metalloenzymes.
While testing our collection of αKG-dependent enzymes for nitrene-transfer activity, we found that one protein, wild-type ethylene-forming enzyme from Pseudomonas savastanoi (PsEFE WT), showed greater than background activity for the aziridination of styrene and tosyl azide (Figure 4-8,
Table 4-1).
Figure 4-8. Nitrene-transfer reaction catalyzed by non-heme iron proteins.
Table 4-1. Activities of α-KG-dependent iron enzymes toward aziridination to form 3.
Enzyme Relative activity
P. savastanoi ethylene-forming enzyme 12.0 Streptomyces sp. 2-aminobutyric acid chlorinase 0.93 A. thaliana anthocyanidin synthase 0.54 G. oxydans leucine dioxygenase 1.11
E. coli taurine dioxygenase 0.61
S. vinaceus arginine hydroxylase 0.57 S. muensis leucine hydroxylase 0.61 Bovine serum albumin (negative control) 1.00
PsEFE WT has been studied both for the potential biotechnological application of efficient ethylene production38 as well as the fact that it has two native activities.39 In its first, more classical αKG-dependent enzyme activity, an equivalent of αKG is used to oxidize L-Arg to form pyrroline-5-carboxylic acid, an intermediate in proline synthesis (Figure 4-9a). Its second native reaction is the conversion of αKG into ethylene (Figure 4-9b).
Figure 4-9. Reactions catalyzed natively by Pseudomonas savastanoi ethylene-forming enzyme (PsEFE). (a) An equivalent of αKG is used to oxidize L-Arg to form pyrroline-5-carboxylic acid, an intermediate in proline synthesis, with guanidine, succinate, carbon dioxide, and water as byproducts. (b) αKG is converted into ethylene with water and three equivalents of carbon dioxide as byproducts.
When testing new reactions, it is critical to ensure that product formation is enzyme catalyzed and not a background reaction. Several additional controls were carried out to ensure that the activity was due to PsEFE WT and not due to background activity. These controls include running the reaction in the absence of key reagents such as iron, ascorbate (reductant), and αKG (co-substrate). In addition, we generated a protein variant which lacked two of the coordinating amino-acid residues (PsEFE H189A D191A). This double mutant protein still solubly expressed and purified, but was expected to have a greatly disrupted metal-binding site. These control reactions showed that omission of iron or the use of the double mutant abolishes activity, indicating that the reaction is enzymatic and occurs in the iron-binding site (Table 4-2). The activity is also proportional to the catalyst loading, lending further evidence that it is enzymatic. Another test is whether the product is enantioenriched; while the enzyme might form racemic product, it is unlikely that a background reaction would form an enantioenriched product. Chiral HPLC analysis showed that PsEFE WT was (R)- selective, with 25% enantiomeric excess (ee); the P411BM3 variant previously engineered for this nitrene-transfer reaction was (S)-selective.
Table 4-2. Control and ligand-substitution reactions for PsEFE WT aziridination to form 3.
Deviation from standard conditions Aziridine yield (%) H189A D191A mutant (disrupted iron binding) <0.01%
No iron 0.01%
No αKG or analog 0.04%
None (standard conditions) 0.08%
Succinate instead of αKG 0.11%
Acetate instead of αKG 0.56%
N-oxalylglycine instead of αKG 0.64%
No ascorbate, acetate instead of αKG 0.50%
Given the ability to catalyze the aziridination of tosyl azide and styrene, we investigated the scope of nitrene-transfer reactions that PsEFE WT could catalyze. The tests focused on the aziridination of unactivated alkenes, which had not been reported with the P411BM3
aziridination platform.40 We also tested 4-ethyl anisole, a model substrate for benzylic C–H amination.41 No products were detected for the intermolecular C–H amination of 4-ethyl
anisole or the aziridination of 1-octene, but the aziridination of allylbenzene and 4-phenyl- 1-butene were detected, with 1.5 to 2-fold higher signal than the background and negative control reactions (Figure 4-10).
Figure 4-10. Aziridination of unactivated alkenes. Reaction conditions: 50 µM PsEFE WT purified protein, 1 mM ferrous ammonium sulfate, 1 mM αKG, 10 mM alkene, 10 mM tosyl azide, 5%
ethanol cosolvent, 2 hours, room temperature.
In addition to testing for further nitrene-transfer reactions, an array of carbene-transfer reactions were tested with PsEFE WT. The formation of the metal-carbenoid intermediate is a crucial prerequisite for the carbene-transfer reaction to occur, and its formation is heavily dependent on the catalyst and the specific diazo compound used as a carbene precursor. Four diazo compounds with varied steric and electronic properties were tested (Figure 4-11).
Xenometallation of PsEFE was also attempted, adding first-row transition metals (Mn, Ni, Co, Cu) in place of Fe in reactions. To limit the combinatorial expansion of test reactions, styrene was used as the alkene in all carbene-transfer reaction attempts. GC-MS traces of organic extracts of these reactions were analyzed for mass spectra corresponding to both carbene transfer to styrene and for dimerization of the diazo molecules; no carbene transfer activity above the negative control background was observed, and no clear evidence of carbene formation (e.g. nitrogen evolution) was noted in the reactions.
Figure 4-11. Attempted carbene transfer reactions. Reaction conditions: 50 µM purified PsEFE WT, 100 µM metal salt, 1 mM α-KG, 1 mM sodium dithionite, 10 mM styrene, 10 mM diazo reagent, 5% ethanol cosolvent. Reactions were analyzed via GC-MS.
Following the initial evaluation of PsEFE WT’s new-to-nature reaction scope, we decided to focus on aziridination of styrene with tosyl azide as our model reaction in a directed evolution campaign to develop PsEFE as a new-to-nature biocatalytic platform. As has been observed countless times, mutations which are found to be beneficial when evolving for one function can also provide the starting activity for other reactions of interest;42 engineering PsEFE for this aziridination model reaction would likely enable access to additional new-to- nature reactions.
4.4.1 Analogs of α-ketoglutarate modulate PsEFE’s aziridination activity
Ethylene-forming enzyme requires α-ketoglutarate for both of its native functions, but the nitrene-transfer reaction should not require an equivalent of the co-substrate to go through the catalytic cycle. I therefore hypothesized that α-ketoglutarate would not be strictly required and could potentially be replaced with other compounds to modulate the activity of PsEFE. This change would be analogous to the ability of cytochrome P411BM3 to catalyze carbene- and nitrene-transfer reactions in the absence of the FAD domain (or, equivalently,
as the heme domain only); while the native oxene transfer reaction required the electron transport chain to be intact, the new-to-nature reaction does not have this strict requirement.
Total activity was improved for both indole alkylation43 and C-H functionalization44 by removing a section of the “unnecessary” domains. There is literature precedent that PsEFE can accept a co-substrate other than αKG: Hausinger and coworkers demonstrated that 2- oxoadipate could be used in place of αKG in the formation of pyrroline-5-carboxylic acid, albeit at a 500-fold loss in activity, and other keto-acids were reported to display no detectable P5C formation.39 I tested PsEFE WT aziridination reactions with αKG substituted with several other carboxylate-containing compounds (Figure 4-12). Most resulted in activity equal to or worse than αKG, but acetate and N-oxalylglycine (NOG) both enhanced aziridination activity greater than five-fold (Table 4-2).
Figure 4-12. αKG and analogs tested for their effect on PsEFE aziridination activity. Acetate and N-oxalylglycine display enhanced activity relative to αKG.
There are several potential advantages for replacing αKG in the reaction. αKG is inexpensive (approximately $1/g from Sigma-Aldrich), but replacing αKG with an even cheaper ligand (e.g. acetate at $0.14/g from Sigma-Aldrich) could decrease reaction costs. More importantly, if PsEFE were engineered to selectively bind another molecule over αKG, it
should prevent the competing native oxene transfer from occurring, even under aerobic conditions. The ability to substitute the ligand by simply changing the reaction conditions and without requiring the synthesis of a non-native metal complex cofactor enables the facile modulation of the primary coordination sphere.