A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase

Article abstract of DOI:10.1073/pnas.1915054117

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

Full text of DOI:10.1073/pnas.1915054117

A de novo peroxidase is also a promiscuous yet
stereoselective carbene transferase
Richard Stennera,b, Jack W. Steventona,c, Annela Seddonb,d, and J. L. Ross Andersona,c,1

aSchool of Biochemistry, University of Bristol, BS8 1TD Bristol, United Kingdom; bBristol Centre for Functional Nanomaterials, HH Wills Physics Laboratory,
University of Bristol, BS8 1TL Bristol, United Kingdom; cBrisSynBio Synthetic Biology Research Centre, University of Bristol, BS8 1TQ Bristol, United Kingdom;
and dSchool of Physics, HH Wills Physics Laboratory, University of Bristol, BS8 1TL Bristol, United Kingdom
Edited by William F. DeGrado, University of California, San Francisco, CA, and approved December 10, 2019 (received for review August 29, 2019)
By constructing an in vivo-assembled, catalytically proficient perox-
idase, C45, we have recently demonstrated the catalytic potential of
simple, de novo-designed heme proteins. Here, we show that C45’s
enzymatic activity extends to the efficient and stereoselective inter-
molecular transfer of carbenes to olefins, heterocycles, aldehydes,
and amines. Not only is this a report of carbene transferase activity
in a completely de novo protein, but also of enzyme-catalyzed ring
expansion of aromatic heterocycles via carbene transfer by any
enzyme.
activities are mostly dependent on accessing hypothetical heme-
based carbene and nitrene intermediates (23) analogous to the
oxene intermediates, observed in the catalytic cycles of heme-
containing peroxidases and oxygenases (18). Several groups have
now reported examples of natural and engineered hemoproteins
that catalyze cyclopropanations (20, 24–31), C–H insertions (32–
35), carbonyl olefinations (36, 37), N–H insertions (21, 38), C–H
amination/amidation (39, 40), boron alkylations (41), aziridinations
(42), and C–Si bond formations (22), most of which have been
exposed to several rounds of directed evolution to improve stereo-
selectivity and product yield. Excluding the aziridation reactions, an
electrophilic metallocarbenoid intermediate (Fig. 1B) is hypothesized
to be responsible for carbene transfer to a suitable nucleophile (e.g.,
olefin) (23, 43) in these reactions. We therefore reasoned that if the
metallocarbenoid intermediate could be detected spectroscopically
in C45, then the de novo enzyme may function as a promiscuous
carbene transferase, catalyzing a range of important and challenging
organic transformations.
de novo protein design enzyme design carbene transfer biocatalytic
|
|
|
ring expansion biocatalysis
|
espite the significant advances in protein design, there still
remain few examples of de novo enzymes constructed from
D
bona fide, de novo protein scaffolds that both approach the
catalytic efficiencies of their natural counterparts and are of
potential use in an industrial or biological context (1–10). This
reflects the inherent complexities experienced in the biomolecular
design process, where approaches are principally focused on either
atomistically precise redesign of natural proteins to stabilize re-
action transition states (1–4) or imprinting the intrinsic chemical
reactivity of cofactors or metal ions on simple, generic protein
scaffolds (5–10); both can be significantly enhanced by imple-
menting powerful, yet randomized, directed evolution strategies
to hone and optimize incipient function (11, 12). While the latter
approach often results in de novo proteins that lack a singular
structure (6, 13, 14), the incorporation of functionally versatile
cofactors, such as heme, is proven to facilitate the design and
construction of de novo proteins and enzymes that recapitulate
the function of natural heme-containing proteins in stable, sim-
ple, and highly mutable tetrahelical chassis (termed maquettes)
(15–17). Since the maquettes are designed from first principles,
they lack any natural evolutionary history, and the associated
functional interdependency that is associated with natural pro-
tein scaffolds can be largely circumvented (15).
Results and Discussion
Generation of Reactive Metallocarbenoids in the De Novo-Designed
C45 and an Engineered Cytochrome c (Rma-TDE). To spectroscopi-
cally isolate the metallocarbenoid intermediate, we rapidly
mixed ferrous C45 with the carbene precursor, ethyl diazoacetate
(EDA), at 5 °C in a stopped-flow spectrophotometer. Concomitant
with the disappearance of the ferrous C45 spectrum was the
appearance of a spectroscopically distinct species over 60 s, with
Significance
While the bottom-up design of enzymes appears to be an in-
tractably complex problem, a minimal approach that combines
elementary, de novo-designed proteins with intrinsically reactive
cofactors offers a simple means to rapidly access sophisticated
catalytic mechanisms. Not only is this method proven in the
reproduction of powerful oxidative chemistry of the natural
peroxidase enzymes, but we show here that it extends to the
efficient, abiological—and often asymmetric—formation of strained
cyclopropane rings, nitrogen–carbon and carbon–carbon bonds, and
the ring expansion of a simple cyclic molecule to form a precursor
for NAD+, a fundamentally important biological cofactor. That the
enzyme also functions in vivo paves the way for its incorporation
into engineered biosynthetic pathways within living organisms.
We have recently reported the design and construction of a
hyperthermostable maquette, C45, that is wholly assembled in
vivo, hijacking the natural Escherichia coli cytochrome c maturation
system to covalently append heme onto the protein backbone (Fig.
1A) (6). The covalently linked heme C of C45 is axially ligated by a
histidine side chain at the proximal site, and it is likely that a water
molecule occupies the distal site, analogous to the ligation state of
natural heme-containing peroxidases (18) and metmyoglobin (19).
Not only does C45 retain the reversible oxygen-binding capability
of its ancestral maquettes (6, 15, 16), but it functions as a pro-
miscuous and catalytically proficient peroxidase, catalyzing the
oxidation of small molecules, redox proteins, and the oxidative
dehalogenation of halogenated phenols with kinetic parameters
that match and even surpass those of natural peroxidases (6).
It has been recently demonstrated that several natural heme-
containing proteins and enzymes [e.g., cytochromes P450 (20),
globins (21), and cytochrome c (22)] are capable of accessing
many chemistries intrinsic to the heme cofactor, not all of which
are essential to life-supporting biological roles. These reported
Author contributions: R.S., J.W.S., A.S., and J.L.R.A. designed research; R.S., J.W.S., and
J.L.R.A. performed research; R.S., J.W.S., A.S., and J.L.R.A. analyzed data; and R.S. and
J.L.R.A. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution License 4.0
(CC BY).
1To whom correspondence may be addressed. Email: ross.anderson@bristol.ac.uk.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1915054117/-/DCSupplemental.
First published January 2, 2020.
www.pnas.org/cgi/doi/10.1073/pnas.1915054117
PNAS
|
January 21, 2020
|
vol. 117
|
no. 3
|
1419–1428

A
C
B
Fig. 1. Metallocarbenoid formation and its reactive potential within a de novo-designed c-type cytochrome maquette, C45. (A) Single snapshot from a 1-μs
molecular-dynamics simulation of the C45 maquette (6). (B) Formation and potential reactivity of a heme-based metallocarbenoid intermediate, illustrating
aldehyde olefination, olefin cyclopropanation, and amine N–H insertion reactions. (C) UV/visible EDA-treated C45 (red), engineered Rma-TDE (green), and
MP-11 (blue) obtained by rapid mixing experiments in a stopped-flow spectrophotometer. The putative metallocarbenoid species was generated by mixing
2.5 mM EDA with 7.5 μM ferrous heme protein in 100 mM KCl and 20 mM CHES, pH 8.6 (10% EtOH).
a red-shifted Soret peak at 429 nm and broad Q bands centered
at 543 and 586 nm (Figs. 1C and 2A). Under these conditions,
this spectrum persisted for 1,000 s with almost no degradation
(Fig. 2B), and it was spectroscopically consistent with spectra
reported for experimentally produced carbene:iron porphyrin
complexes (44). For our proposed C45 metallocarbenoid inter-
mediate, the cold conditions on rapid mixing proved essential to
spectroscopic observation, as room-temperature experiments
resulted in spectra indicative of carbene-induced heme degra-
dation, consistent with a mechanism proposed by Arnold and
colleagues (45). It was also necessary to employ an ethanol:water
mixture to ensure EDA solubility and stability for generating the
putative intermediate in the stopped flow at 5 °C, and we ob-
served an identical, long-lived spectrum at ethanol concentra-
tions between 10 and 50% (SI Appendix, Fig. S1). While ethanol
concentrations above 50% are generally avoided in buffered
protein solutions due to denaturation, small helical bundles such
as C45 can readily tolerate such aqueous:organic mixtures (5, 6),
retaining structure and catalytic activity. Substituting EDA for
benzyl-diazoacetate (BnDA) and tert-butyl-diazoacetate (tBuDA)
also resulted in the appearance of intermediates with near-identical
spectroscopic properties to the EDA-generated C45 species (Fig.
2C), demonstrating the intrinsic flexibility of the active site in
accommodating bulky diazoacetate substituents. We were sub-
sequently able to generate and measure mass spectra of these
putative intermediates using mild ionization/near-native conditions
using positive electrospray ionization mass spectrometry (ESI-MS)
(SI Appendix, Fig. S2). Mass spectra of the EDA-, BnDA-, and
tBuDA-generated metallocarbenoids of C45 exhibited the mass
differences expected for the adducts, further confirming the
nature of the species generated under these conditions.
lower temperature, we postulate that the reported ultraviolet
(UV)/visible spectra instead represent an alternative, yet currently
unidentified, species. In contrast to C45, Rma-TDE exhibits a lag
phase during the formation of the putative intermediate, with
noticeably slower kinetics compared to C45 under the same ex-
perimental conditions. To probe this further, we measured the
EDA concentration-dependent kinetics of the putative metallo-
carbenoid formation for C45 and Rma-TDE (Fig. 2D and SI
Appendix, Fig. S3). C45 exhibited both a higher limiting rate
constant (klim
) for metallocarbenoid formation and lower
pseudo-Michaelis constant (K1) for EDA (klim = 0.50 s−1; K1 =
74 μM) compared to Rma-TDE (klim = 0.14 s−1; K1 = 490 μM),
demonstrating that not only does C45 bind EDA more rapidly
than Rma-TDE, but it likely has a significantly higher affinity for
the carbene precursor.
Cyclopropanation Activity of C45 and Rma-TDE. We subsequently
investigated the ability of C45 to act as an active carbene
transferase in the cyclopropanation of styrene, commonly used as
an acceptor for heme protein-derived metallocarbenoids (20).
To confirm the identity of the spectroscopically isolated species
as the putative metallocarbenoid intermediate, we rapidly mixed
ferrous C45 with 100 μM EDA and 3 mM styrene at 5 °C in a
stopped-flow spectrophotometer (Fig. 3). Under these conditions,
the same putative intermediate spectrum appeared over 60 s, but
then decayed slowly to the starting ferrous C45 spectrum, con-
sistent with the proposed mechanism of heme-catalyzed carbene
transfer to the olefin, in which there is no net transfer of elec-
trons from the heme (47, 48). No other spectroscopically distinct
species was observed during this experiment. The putative metallo-
carbenoid species of the engineered Rma-TDE displayed
analogous behavior to C45 in the presence of styrene, with the
disappearance of the metallocarbenoid intermediate and con-
comitant reappearance of the ferrous Rma-TDE spectrum (Fig.
3). In contrast, the aerobically generated species prepared by
room-temperature mixing of Rma-TDE with EDA did not exhibit
such behavior, and there was no detectable cyclopropanation ac-
Under identical conditions to C45, we individually mixed ferrous
microperoxidase-11 (MP-11) and an engineered cytochrome c from
Rhodothermus marinus (46) (Rma-TDE) with EDA, the latter with
an established carbene transferase activity with respect to silanes
(46). Near-identical spectra to the putative metallocarbenoid
C45 were obtained (Fig. 1C), though significant differences were
apparent between our spectra and those reported for the metal- tivity. Given these data, we assigned the spectroscopically distinct
locarbenoid complex of Rma-TDE (46). Since our spectra were species we described above to the C45 and Rma-TDE metallo-
collected under more rigorously anaerobic conditions than the
previous study (stopped-flow spectrophotometer housed in an
anaerobic glove box; <5 parts per million [ppm] O2) and at a
carbenoid intermediates. It should be noted that, at this time, we
cannot definitively assign these spectra as either the nonbridging
metallocarbenoid species observed by Lewis et al. (46) or the
1420
|
www.pnas.org/cgi/doi/10.1073/pnas.1915054117
Stenner et al.

A
B
C
D
Fig. 2. Stability and reactivity of the metallocarbenoid:C45 intermediate. (A) Time course of electronic spectra recorded following rapid mixing of ferrous
C45 (red) with EDA in 10% EtOH at 5 °C. The appearance of the metallocarbenoid intermediate (blue) is concomitant with the disappearance of the ferrous
C45 spectrum. Spectra presented were recorded 1, 2, 4, 6, 8, 10, 15, 20, 50, and 100 s after mixing. (B) Metallocarbenoid formation and stability in the absence
of styrene substrate. Single-wavelength traces represent the time course of ferrous C45 (418 nm; blue; 7.5 μM protein, 10% EtOH) and metallocarbenoid:C45
adduct (437 nm; red) following rapid mixing of ferrous C45 with 500 μM EDA at 5 °C. Once formed, the metallocarbenoid:C45 adduct persists for the duration
of the experiment (1,000 s). (C) Electronic spectra of metallocarbenoid intermediates formed between ferrous C45 and EDA, BnDA, and tBuDA following rapid
mixing in the stopped-flow apparatus. (D) EDA-concentration-dependent formation of the C45 metallocarbenoid adduct. Kinetic data were recorded by using
a stopped-flow spectrophotometer and analyzed as described in Experimental Details. The limiting rate constant (klim) and pseudo-Michaelis constant (K1) for
metallocarbenoid formation are 0.50 s−1 and 74 μM, respectively. Data were collected in triplicate, and error bars represent the SD.
porphyrin-bridging species observed by Hayashi et al. (49) in the analysis of the C45-catalyzed reaction revealed the presence
crystal structures of engineered cytochrome c or N-methylhistidine– of 2 peaks in the chiral chromatogram at 7.23 and 9.00 min,
ligated myoglobin variant [Mb(H64V,V68A)], respectively. How-
ever, given the identical nature of the C45 and Rma-TDE
corresponding to the (R,R) and (S,S) enantiomers of the ethyl
2-phenylcyclopropane-1-carboxylate (Et-CPC; 1a) product (SI
spectra, it would seem likely that the spectra obtained represent Appendix, Figs. S4 and S5). Following base hydrolysis of the
the nonbridging ligation observed in the Rma-TDE Me-EDA crystal Et-CPC to the corresponding acid (2-phenylcyclopropane-1-
structure (46). Additionally, while the rate of metallocarbenoid
intermediate and subsequent product formation appeared relatively
low in our stopped-flow experiments, it is worth noting that the
selected conditions were necessary for maximizing the quantity
of intermediate in the stopped-flow apparatus and that subsequent
activity assays were carried out at higher substrate concentrations
(both EDA and styrene), higher temperature, and lower ethanol
concentrations. This would undoubtedly lead to higher reaction
rates than those presented in the stopped-flow data here.
carboxylic acid; CPC) and further LC-MS and chiral HPLC
analysis against commercial standards (SI Appendix, Figs. S5 and
S6), we determined that the C45-catalyzed cyclopropanation of
styrene was highly diastereoselective (>99% de), occurred at high
yield (80.2%, total turnover number [TTN] = 802, turnover fre-
quency [TOF] = 6.68 min−1), and exhibited significant enantio-
selectivity, with an enantiomeric excess (ee) of 77% in favor of the
(R,R) enantiomer. C45 will accept derivatized diazoacetates and
para-substituted styrenes as substrates for cyclopropanation, pro-
Cyclopropanation reactions were initiated at 5 °C under an- ducing products with varying stereoselectivities and yields (1a–h, 2a,
aerobic conditions and at low ethanol concentrations (5% EtOH) and 2b; Fig. 4, Table 1, and SI Appendix, Figs. S7–S9). In particular,
and were subsequently allowed to warm to room temperature. We C45/EDA-catalyzed cyclopropanation of p-trifluoromethylstyrene,
then analyzed the cyclopropanation activity of C45 and hemin, p-methoxystyrene, and p-fluorostyrene occurred with exceptional
respectively, when mixed with EDA and styrene by chiral high- enantioselectivity for the (R,R) product (99.6% ee for F3C-
performance liquid chromatography (HPLC) and liquid chroma- substituted, 1d; 92.8% ee for MeO-substituted, 1e; and 96.6%
t
tography–mass spectrometry (LC-MS). Hemin exhibited little ee for F-substituted, 1h). However, BuDA and BnDA provided
cyclopropanation activity, and, under the reaction conditions
lower yields and enantioselectivities of products 2a (18.5% and
employed here, only unreacted EDA was observed. However, 40.6% ee) and 2b (19.5% and 66.8% ee). We postulate that the
Stenner et al.
PNAS
|
January 21, 2020
|
vol. 117
|
no. 3
|
1421

C45 + EDA + Styrene
Carbene adduct then
product formation
A
C
E
415 nm
444 nm 520 nm
550 nm
416 nm
429 nm
C45
0.50
0.45
0.40
0.35
0.30
0.25
418 nm
1 sec
66 secs
1000 secs
(Fe2+
)
AbsDecay
5 x 10-3 OD
(1000 - 66 secs)
550 nm
586 nm
AbsAdduct
437 nm
(Metallocarbenoid)
0.05 OD
(0 - 66 secs)
0.05 OD
400
450
500
550
600
650
0
400
800
400
450
500
550
600
650
Wavelength (nm)
Time (secs)
Wavelength (nm)
B
D
F
416 nm
Rma-TDE + EDA + Styrene
Carbene adduct then
416 nm
429 nm
522 nm
552 nm
product formation
0.7
0.6
0.5
0.4
0.3
0.2
Rma-TDE
1 sec
233 secs
600 secs
418 nm
AbsDecay
0.01 OD
(Fe2+
)
(1000 - 100 secs)
586 nm
552 nm
AbsAdduct
0.1 OD
433 nm
(Metallocarbenoid)
0.1 OD
(0 - 100 secs)
400
450
500
550
600
650
0
200
400
600
400
450
500
550
600
Wavelength (nm)
Time (secs)
Wavelength (nm)
Fig. 3. Reactivity of the metallocarbenoid intermediates of C45 and Rma-TDE. (A and B) Electronic spectra of C45 (A) and Rma-TDE (B) recorded after rapid
mixing of ferrous heme protein (7.5 μM; red trace) with EDA (500 μM) and styrene (3 mM) at 5 °C in the stopped-flow spectrophotometer. Green traces
correspond to spectra recorded at time points where the maximum quantity of metallocarbenoid was accumulated; blue traces correspond to the final spectra
recorded in the experiments. (C and D) Metallocarbenoid formation and decay of C45 (C) and Rma-TDE (D) in the presence of styrene. Single-wavelength
traces represent the time course of ferrous heme protein (blue traces) (7.5 μM protein) and metallocarbenoid adduct (red) following rapid mixing of ferrous
heme protein with 500 μM EDA and 3 mM styrene at 5 °C. (E and F) Electronic difference spectra highlighting the spectroscopic changes associated with
metallocarbenoid formation and decay for C45 (E) and Rma-TDE (F). The lower, red traces demonstrate the spectroscopic changes that occur during the
formation of the metallocarbenoid, which are identical to those observed during its subsequent decay (upper, blue traces). These indicate reformation of the
initial ferrous species after carbene insertion and after EDA is exhausted.
observed stereoselectivity exhibited by C45 resulted from the
intrinsic asymmetry of the protein scaffold, likely through spe-
variant of C45 (AP3.2) with switched and remarkably high
enantioselectivity toward the (S,S) product (ee = 99%), albeit
cific positioning of the reactive carbene adduct, as proposed by with a lower yield (41.5%) compared to C45 (SI Appendix, Fig.
S10). To probe the flipped stereoselectivity and reduced yield
exhibited by AP3.2, we purified and characterized the protein
both spectroscopically and functionally. AP3.2 exhibited near-
identical UV/visible ferric and ferrous spectra to C45 (6) (SI
Appendix, Fig. S11A) and a circular dichroism (CD) spectrum
consistent with that expected for a 4-helix bundle maquette (6,
15, 17) (SI Appendix, Fig. S11B), albeit with reduced secondary
structure compared to C45. The 8 mutations to C45 (F11Y/
G39S/D48Y/F53S/F83S/G109A/F132S/E133G) destabilized the
protein scaffold, with AP3.2 exhibiting a 37 °C drop in the
thermal melting transition (Tm = 49 °C) in comparison to C45 (6)
(SI Appendix, Fig. S11C). Given that 3 of these substitutions
replaced core, hydrophobic phenylalanines in helical positions
with the hydrophilic serine, this was not surprising, especially
given that serine has a significantly lower helical propensity than
phenylalanine (50). Mapping the AP3.2 mutations onto our
computationally generated C45 model (6) (SI Appendix, Fig.
Villarino et al. (25) and more recently observed in the crystal
structure of the methyl-EDA adduct of the engineered cyto-
chrome c (46). Our previous work (6) has demonstrated that C45
exists in a dynamic and conformationally heterogeneous state, at
least under the conditions and concentrations used for NMR stud-
ies. This suggests that a native-like state is neither required for this
type of chemical catalysis in a protein nor for achieving a high degree
of stereoselectivity in the metalloprotein-catalyzed cyclopropanation
of styrene. Indeed, a conformationally dynamic environment at the
distal face of the heme may facilitate access of both the diazoacetate
and styrene (46) into the active site, while subsequently providing
sufficient space for the olefin to asymmetrically attack the
electrophilic metallocarbenoid.
Altering the Stereoselectivity of Styrene Cyclopropanation in C45.
The apparent asymmetry provided by the protein scaffold offers
a means by which we can improve subsequent C45-derived ma-
quettes. In fact, small-scale screening of a C45 library generated S11D) did not immediately reveal an obvious explanation for the
by successive generations of error-prone PCR (epPCR) and
originally created for improved peroxidase activity revealed a
pronounced change in stereoselectivity we observed, as only the
F53S mutation was positioned near to the carbene binding site at
1422
|
www.pnas.org/cgi/doi/10.1073/pnas.1915054117
Stenner et al.

A
B
C
Fig. 4. Carbene transferase activity of C45. (A) Cyclopropanation of substituted styrenes catalyzed by C45. TTNs and % ee for each combination of ferrous
C45 with para-substituted styrenes and functionalized diazoacetates are shown. Only the (R,R) cyclopropanated product is displayed in the reaction scheme,
representing the dominant product in all cases. All reactions were carried out with 0.1% catalyst loading (10 μM C45) at the following concentrations of
reagents: 10 mM sodium dithionite, 10 mM diazo compound, and 30 mM substituted styrene in 100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH/H2O. (B) N–H
insertion of primary and secondary amines catalyzed by C45. Only the monofunctionalized product of the p-chloroaniline insertion reaction is shown, and the
corresponding TTN is calculated based on the yield of both monosubstituted and disubstituted products. All reactions were carried out with 0.1% catalyst
loading (10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 10 mM EDA, and 30 mM amine substrate in 100 mM KCl, 20 mM
CHES (pH 8.6), and 5% EtOH/H2O. (C) Olefination of substituted benzaldehydes catalyzed by C45. All reactions were carried out with 0.1% catalyst loading
(10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 10 mM PPh3, 10 mM EDA, and 30 mM substituted benzaldehyde in
100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH/H2O.
the distal face of the heme. To examine reactivity in more detail,
we generated the AP3.2 metallocarbenoid intermediate by rapid
mixing under the same conditions as described for C45, and we
observed an identical intermediate spectrum that accumulated
on a comparable timescale to C45 (SI Appendix, Fig. S12) and, in
the presence of styrene, decayed at a comparable rate to C45
with identical associated spectroscopic changes (SI Appendix,
Fig. S13 A–C). To test the possibility of an electronic effect
playing a role in the suppressed activity of AP3.2, we used redox
potentiometry to measure the AP3.2 heme redox potential (Em)
(SI Appendix, Fig. S13D). AP3.2 exhibited a 26-mV negative shift
in redox potential with respect to C45 [C45, Em = −176 mV (6);
AP3.2, Em = −202 mV], indicating slight stabilization of the
ferric form, possibly due to increased solvent accessibility at the
heme (51). While this shift also indicated that the extent of
electron donation to the bound carbene was indeed altered fol-
lowing the mutations, it was relatively modest in comparison to
those observed after heme axial ligand substitution in engineered
P450s (51, 52). In these cases, the correlation between large re-
dox potential changes and the specific reactivity of the metallocarbenoid
Stenner et al.
PNAS
|
January 21, 2020
|
vol. 117
|
no. 3
|
1423

Table 1. C45-catalyzed cyclopropanation of styrene and para-substituted styrenes by EDA and of styrene by
substituted diazoacetates
Product
R1
R2 % Yield (R,R) % Yield (S,S)
de (E)%
ee (R,R)% TTN (R,R) Reaction time (min) TOF/min−1
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
H
OH
Cl
F3C
OMe Et
CN
tBu
F
H
H
Et
Et
Et
Et
80.2 8.0
36.5 5.7
50.7 3.3
79.7 4.0
45.5 1.7
55.0 8.7
56.7 1.0
47.0 0.8
18.5 8.1
19.5 1.4
10.4 1.80
2.65 0.67
5.81 0.19
0.14 0.04
1.71 0.10
4.75 0.92
5.52 0.12
0.83 0.01
7.00 1.54
4.00 1.41
99.9 0.1 77.0 2.8
99.9 0.1 86.6 1.3
99.9 0.1 79.4 0.6
99.9 0.1 99.6 0.1
99.9 0.1 92.8 0.2
99.9 0.1 84.2 0.5
99.9 0.1 82.3 0.4
99.9 0.1 96.6 0.2
99.9 0.1 40.6 16.5
99.9 0.1 66.8 7.4
802
365
507
797
455
550
567
470
185
195
120
120
120
120
120
120
120
120
120
120
6.68
3.04
4.23
6.64
3.80
4.58
4.73
3.92
1.92
2.09
Et
Et
Et
tBu
Bn
Values are SDs.
is currently unclear, at least experimentally, and increased re-
activity resulting from a more electrophilic carbene adduct would
be expected to correlate with a more electron-withdrawing,
higher-redox potential heme (43). Furthermore, we identified
TOF = 7.99 min−1) as per the literature (26) and Rma-TDE exhib-
iting 70.6% ee for the (R,R) product (yield = 73.5%, TTN = 735,
TOF = 6.12 min−1). While the yield from the C45-catalyzed reaction
(80.2%) did not surpass that of Mb(H64V,V68A), it is among
another C45 variant (APR1) from the library with similarly altered the highest yields observed for the catalytic cyclopropanation of
redox potential (APR1; Em = −197 mV; ΔEm = −21 mV), but styrene by EDA, surpassing those exhibited by almost all reported
with negligible difference in yield or enantioselectivity [yield = P450 (20, 26–30) and LmrR (25) variants, as well as by several
85.6%, TTN = 856, ee(R,R) = 78.0%; SI Appendix, Fig. S14],
engineered myoglobins (24, 26, 30). In addition, Rma-TDE
therefore highlighting that such small differences in Em likely exhibited a relatively high yield and enantioselectivity for sty-
contribute little to the overall yield of product under these re- rene cyclopropanation, despite being engineered for carbene
action conditions. As is the case for many enzymes improved by insertion into Si–H bonds.
random laboratory evolution methodologies (52), it is therefore
N–H Insertion and Carbonyl Olefination Reactions Catalyzed by C45.
With stereoselective carbene transferase activity firmly established
for the cyclopropanation of styrene by C45, we explored the sub-
strate promiscuity of C45 toward similar reactions resulting in N–H
insertions and carbonyl olefinations. Following reaction conditions
identical to those described above for cyclopropanation and using
HPLC and LC-MS to identify insertion products, we observed the
C45-catalyzed insertion of EDA/tBuDA/BnDA-derived carbene
into the N–H bonds of piperidine and para-chloroaniline (3a–c and
not unambiguously clear why this altered reactivity and enantio-
selectivity occurs, and a currently uncharacterized combination of
static and dynamic processes likely play a role in the observed
catalytic and stereoselective changes. It does, however, demon-
strate well the mutability and catalytic potential of the maquette
scaffold and will be explored in more depth in future work.
Benchmarking the Cyclopropanation Activity of C45. Since there are
now several examples of engineered and natural proteins capable
of catalyzing carbene transfer, we wished to benchmark the ac- 4a–c; Fig. 4, Table 3, and SI Appendix, Figs. S17–S21), with single-
tivity of C45 against some notable and well-characterized carbene and double-insertion products resulting from carbene transfer to
transferases. To this end, we directly compared the C45-catalyzed the primary amine of para-chloroaniline when EDA was employed.
cyclopropanation of styrene with EDA against the corresponding Notably, C45 achieved high yields for the insertion of BnDA-
reactions with Rma-TDE (22, 46) and a double mutant of sperm derived carbene into the piperidine and para-chloroaniline N–H
whale myoglobin, Mb(H64V,V68A) (26) (Table 2 and SI Appen- bonds, with 88.3% and 89.4%, respectively (TTN = 883 for 3c;
dix, Figs. S15 and S16). While Rma-TDE was engineered toward TTN = 894 for 4c). For carbonyl olefination, the addition of
silane alkylations (22), it forms a structurally characterized carbene
triphenylphosphine to the reaction mixture is necessary to facilitate
adduct and is a c-type cytochrome, rendering it the most spectro- the formation of a ylide that, through the generation of an
scopically similar carbene transferase to C45 in the literature. In
contrast, the cyclopropanation activity of Mb(H64V,V68A) and
other Mb variants toward styrene has been extensively studied and
is both high yielding and highly enantioselective for the (S,S)
product (26). Under identical conditions to the reactions described
above for C45, we confirmed that both proteins catalyze the
oxaphosphetane intermediate, spontaneously collapses to the product
(36, 37). We investigated the olefination of benzaldehyde and 3
substituted benzaldehyde variants with differing electron-donating and
-withdrawing properties: p-nitrobenzaldehyde, p-cyanobenzaldehyde,
and 3,4,5-trimethoxy-benzaldehyde (5a–d). Reactions were
performed for 2 h, after which the products were again analyzed
cyclopropanation of styrene, with Mb(H64V,V68A) exhibit- by HPLC and LC-MS, and though yields and TTNs were typically
ing >99% ee for the (S,S) product (yield = 95.9%, TTN = 959, low, they were consistent with values reported for several myoglobin
Table 2. Comparison of enzyme-catalyzed cyclopropanations of styrene by EDA
Enzyme
R1 R2 % Yield (R,R) % Yield (S,S)
de (E)%
ee (S,S)% ee (R,R)%
TTN*
Reaction time, min TOF/min−1
C45
H
H
H
H
H
Et
Et
Et
Et
Et
80.2 8.0
10.4 1.80 99.9 0.1
ND
99.9 0.1 99.9 0.1
99.9 0.1 ND
99.9 0.1 99.9 0.1
99.9 0.1 ND
77.0 2.8
ND
70.6 2.0 735 (R,R)
ND 415 (S,S)
78.0 8.5 856 (R,R)
802
959 (S,S)
120
120
120
120
120
6.68
7.99
6.12
3.45
7.13
MgB (H64V/V68A)
Rma-TDE
AP3.2
0
95.9 0.5
12.6 1.0
41.5 4.6
11.04 4.8
73.5 5.7
0
85.6 4.3
APR1
Values are SDs. ND, not detected.
*Indicates the TTN for the specific defined enantiomeric species.
1424
|
www.pnas.org/cgi/doi/10.1073/pnas.1915054117
Stenner et al.

Table 3. C45-catalyzed N-H insertion reactions
Product
Substrate
R
% Yield (single)
% Yield (double)
Single/double
TTN
Reaction time, min
TOF/min−1
3a
3b
3c
4a
4b
4c
Piperidine
Piperidine
Piperidine
p-chloroaniline
p-chloroaniline
p-chloroaniline
Et
tBu
Bn
Et
tBu
Bn
78.9 11.9
62.1 8.9
88.3 13.1
67.6 0.3
72.5 0.2
89.4 0.3
n/a
n/a
n/a
n/a
n/a
n/a
12.7:1
0
789
621
883
676*
725
894
120
120
120
120
120
120
7.99
5.17
7.36
5.63
6.04
7.45
5.21 0.3
0
0
0
Values are SDs. n/a, not applicable.
*Indicates that this TTN corresponds solely to the single insertion product.
variants (Fig. 4 and SI Appendix, Figs. S22–S24 and Table S1)
(37). Interestingly, C45 did not exhibit discernible diastereoselectivity
for carbonyl olefination, possibly indicating that the ylide is released
from the protein prior to rearrangement and final product formation.
Nevertheless, the ability to catalyze these reactions further illustrates
the utility of C45 as a general carbene transferase enzyme.
6 to produce the cyclopropane-containing bicyclic system 8 sus-
ceptible to spontaneous ring opening and rearomatization (Fig.
5A). Using the same reaction conditions as those described for
C45-catalyzed styrene cyclopropanation, we investigated whether
C45 could catalyze the ring expansion of pyrrole (6) with ethyl-2-
bromo-2-diazoacetate (7) (57) as the carbene precursor. After 2 h,
the reaction was analyzed by using HPLC and LC-MS, confirming
the production of the ring-expanded product, ethyl nicotinate (9a)
at 69.4% yield (Fig. 5B and SI Appendix, Figs. S25 and S26). Sub-
sequent base-catalyzed hydrolysis of the product yielded the nicotin-
amide and NAD(P)H precursor niacin (9b), raising the possibility of
engineering a life-sustaining, artificial biochemical pathway from
pyrrole to nicotinamide reliant on the in vivo activity f C45 or a related
de novo-designed enzyme. This is an example of an enzyme—
natural, engineered, or de novo—that is capable of catalyzing ho-
mologous ring expansion reactions via a carbene transfer mechanism.
C45-Catalyzed Ring Insertion In Vitro and In Vivo. Ring-expansion
reactions are exceptionally useful in organic synthesis, as they
provide a reliable and facile method for acquiring large, expanded
ring systems (53, 54). Though currently underused, the homolo-
gous ring expansion of nitrogen-containing heteroaromatics could
be of considerable use in the synthesis of pharmaceuticals and
natural products. While both the Buchner ring expansion (55) and
Ciamician–Dennstedt reaction (56) proceed via a cyclopropane-
containing bicyclic system that is subsequently ring-opened, the
ring opening during the latter reaction is facilitated by the ex-
pulsion of a halogen-leaving group. We therefore hypothesized
A Possible Role for C45 in an Engineered NAD+ Biosynthetic Pathway.
that with the halogen-substituted carbene precursor 7, C45 would To further explore the possibility of employing C45 in an es-
catalyze the cyclopropanation of the nitrogen-containing heteroaromatic
sential and life-sustaining pathway from pyrrole to the pyridine
A
B
Fig. 5. Heteroaromatic ring expansion catalyzed by C45. (A) Reaction scheme for the ring-expansion strategy using ethyl 2-bromo-2-diazoacetate, pyrrole,
and ferrous C45. Following carbene transfer to the pyrrole, spontaneous rearrangement of the bicyclic ring system leads to elimination of HBr and formation
of a 6-membered pyridine ring. (B) C18 reversed-phase HPLC traces of the C45-catalyzed ring expansion of pyrrole to ethyl nicotinate. Traces 1 and 2 show the
C45-catalyzed ring expansion compared to a partially hydrolyzed commercial standard of ethyl nicotinate. The ring expansion was carried out with 1%
catalyst loading (10 μM C45) at the following concentrations of reagents: 10 mM sodium dithionite, 1 mM ethyl 2-bromo-2-diazoacetate, and 10 mM pyrrole
in 100 mM KCl, 20 mM CHES (pH 8.6), and 5% EtOH. Traces 3 to 5 show the results of incubating whole cells containing the C45 expression vector and pEC86
harboring the E. coli cytochrome c maturation apparatus. Traces 3 and 4 represent reactions between whole cells, pyrrole, and ethyl 2-bromo-2-diazoacetate
at 3 and 6 h after inoculation and in the absence of the inducer, IPTG. Trace 5 represents the reaction with C45-expressing whole cells, pyrrole, and ethyl 2-
bromo-2-diazoacetate. In this case, the cells were grown for 3 h and induced with 1 mM IPTG, and C45 was expressed for a further 3 h prior to use in the
whole-cell transformation. Reaction conditions are fully described in Experimental Details.
Stenner et al.
PNAS
|
January 21, 2020
|
vol. 117
|
no. 3
|
1425

nucleotides, we examined the ring expansion of pyrrole to ethyl
nicotinate in living E. coli cells. While the E. coli periplasm is
known to be sufficiently oxidizing to promote the effective for-
mation of disulfide bonds, its measured redox potential is
−165 mV (58), which is sufficiently low to ensure that a large
fraction of C45 would be in the active, ferrous state (6). Using an
established procedure for carbene transferase activity under such
conditions (27–29), we tested the ability of E. coli bearing the
C45-expression plasmid and pEC86 (harboring the c-type cyto-
chrome maturation apparatus) (59) to perform the ring-expansion
reaction of pyrrole with ethyl-2-bromo-2-diazoacetate under an-
aerobic conditions (Fig. 5B). Since the maturation apparatus is
constitutively expressed and results in the production of several
heme-containing membrane proteins (CcmC, CcmE, and CcmF)
(60, 61), it was vital to establish whether any intrinsic ring-expansion
activity was detectable in the presence of these proteins. Whole
cells that had been grown for 3 and 6 h in the absence of the
inducer, isopropyl-β-D-1-thiogalactopyranoside (IPTG), had barely
detectable ring-expansion activity, as determined by C18-HPLC
analysis (Fig. 5B). In contrast, cells that were grown for 3 h, in-
duced with IPTG, and grown for a further 3 h displayed significant
ethyl nicotinate formation with a total yield of 76% (Fig. 5B),
indicating that, under these conditions and in vivo, C45 exhibits
the catalytic ring-expansion activity. It also indicates that both
pyrrole and ethyl-2-bromo-2-diazoacetate are able to cross the
outer membrane and access the periplasmically located C45. In-
terestingly, despite the production of ethyl nicotinate by the cells,
we did not observe an increase in the quantity of niacin in the
extract, indicating that E. coli possibly lacks—or does not produce
an appreciable quantity of—an endogenous periplasmic esterase
capable of efficiently hydrolyzing the product. We therefore tested
a recombinant Bacillus subtilis esterase (62) expressed in, and
purified from, E. coli for ethyl nicotinate hydrolysis activity. Incuba-
tion of this esterase with ethyl nicotinate under near-physiological
conditions resulted in the production of niacin (9b; SI Appendix, Fig.
S27), thus highlighting another functional part in a potential bio-
synthetic pathway from pyrrole to the pyridine nucleotides through
niacin.
To this end, we speculate that it is possible to make C45 and the
Bacillus esterase essential to NAD+ biosynthesis. E. coli synthe-
sizes NAD+ through 2 pathways that both involve the production
of NaMN (nicotinic acid mononucleotide) (SI Appendix, Fig. S28),
then NaAD (nicotinate adenine dinucleotide) prior to NAD+
formation (63–66): the de novo pathway, using L-aspartate as a
starting material to produce quinolate, which is a NaMN precursor
(64); or the pyridine ring salvage pathway, where exogenous niacin
or nicotinamide is utilized instead as an NaMN precursor, repre-
senting the favored pathway when niacin and nicotinamide are
abundant in the environment (66). Knocking out a key enzyme in the
aspartate pathway—e.g., nicotinate-nucleotide diphosphorylase—will
provide an auxotrophic strain of E. coli that, when expressing
both C45 and a suitable periplasmically directed esterase [using a
signal sequence such as malE as for C45 (6)] in nicotinamide and
niacin-lacking media, may be capable of converting pyrrole and
ethyl-2-bromo-2-diazoacetate to niacin and, ultimately, NAD+,
recovering the deleterious phenotype. Currently, production of
such an E. coli strain is outside the scope of this study, but this
strategy highlights the mechanism by which we can tractably and
rationally engineer the metabolism of a bacterium to rely on a de
novo enzyme to sustain an essential pathway.
only the high-yielding cyclopropanation of styrene and its de-
rivatives, but also extends to the insertion of carbenes into N–H
bonds, the olefination of carbenes, and a description of enzyme-
catalyzed ring expansion of a nitrogen heterocycle. This dem-
onstrates that abiological function is intrinsic to these de novo-
designed maquettes and that the higher complexity of natural
protein frameworks, such as the globins and cytochromes P450,
is not necessary to support reactivity of this type. Given the altered
product profile exhibited by AP3.2, we have also demonstrated the
flexibility of the maquette framework toward substantial muta-
tional change, profoundly affecting the stereoselectivity of the de
novo enzyme. This indicates that further yield enhancements and
alternative stereoselectivities for these types of reactions are un-
doubtedly achievable through directed evolution, consistent with
the studies reported for the maquette’s natural counterparts (20,
22, 32). Furthermore, there are many beneficial features of C45
that compare favorably against the work described by others with
natural enzymes: C45 is expressed at high levels in E. coli and is
functionally assembled in vivo (6), eliminating the requirement for
the in vitro incorporation of abiological metalloporphyrins (32)
and facilitating in vivo function (27); C45 is thermally stable and
displays excellent tolerance in organic solvents (6), thereby facilitating
its use as a homogenous catalyst in aqueous:organic mixtures. In
conclusion, this work demonstrates the biocatalytic potential and
reactive promiscuity that maquettes possess, and future work will
be concerned with improving the substrate scope and catalytic
performance of these novel, de novo enzymes. It also reinforces
our previous work (6), demonstrating that maquettes and related
de novo proteins are much more than just laboratory curiosities or
ornaments and are, instead, powerful, green catalysts that can play
a valuable role in facilitating challenging organic transformations.
Experimental Details
Detailed experimental methods are provided in SI Appendix.
General and Molecular Biology, Protein Expression, and Purification. All chemicals
were purchased from either Sigma or Fisher Scientific, and cloned synthetic
genes for Rma-TDE and Mb(H64V/V68A) were purchased from Eurofins Ge-
nomics. Protein expression and purification in E. coli T7 Express (NEB) from the
pSHT (for periplasmic expression) or pET45b(+) [for cytoplasmic expression of
Mb(H64V/V68A)] vectors were carried out as described (6). epPCR libraries of
C45, with a mutation rate of 2- to 3-amino-acid mutations, were produced by
using the GeneMorph II EZClone Domain Mutagenesis Kit. The mutation rate
was determined by randomly selecting and sequencing 10 colonies after
transforming the PCR products into E. coli T7 Express cells. This mutation rate
was achieved with 18.7 ng of target DNA (C45 coding sequence). AP3.2 and
APR1 were randomly selected variants from the epPCR C45 library. Each variant
was expressed and purified as described for C45 (6), and the purified proteins
were used in the cyclopropanation assays.
Stopped-Flow Spectrophotometry. Stopped-flow kinetics were conducted by
using an SX20 stopped-flow spectrophotometer (Applied Photophysics)
housed in an anaerobic glove box under N2 ([O2] < 5 ppm; Belle Technology).
In the initial experiments, a solution containing a known concentration of
reduced C45 or AP3.2 [15 μM, 100 mM KCl, and 20 mM 2-(cyclohexylamino)
ethane sulfonic acid (CHES), pH 8.6, reduced with a stoichiometric quantity
of Na2S2O4] was placed in 1 syringe, and a 1 to 5 mM solution of a carbene
t
precursor (either EDA, BuDA, or BnDA) in ethanol (20 to 100%) was placed
in the 2nd syringe. Fifty microliters from each syringe was simultaneously
injected into a mixing chamber, and the progression of the reaction was
monitored spectroscopically at 5 °C and 25 °C, over the course of 180 to
1,000 s, to examine the metallocarbenoid formation and porphyrin degra-
dation pathway, respectively. The formation of the metallocarbenoid was
monitored by following the time-course profiles at 417 nm and 428 to 437 nm,
the Soret peak for reduced C45/AP3.2/Rma-TDE, and the metallocarbenoid
intermediate, respectively. Final concentrations were 7.5 μM ferrous C45/
AP3.2/Rma-TDE and 0.5 to 2.5 mM EDA/tBuDA/BnDA (10 to 50% ethanol).
The kinetics of the formation of the metallocarbenoid for C45/Rma-TDE
were determined by using the same conditions outlined above but using
varying concentrations of EDA (50 μM to 1.5 mM). The rate constants were
calculated at varying substrate concentrations and plotted, and then the
Conclusions
With this work, we have showcased the exceptionally diverse
functionality available in this simple, de novo-designed heme
protein and demonstrated that the carbene transferase activity
intrinsic to the scaffold compares very favorably to that reported
for most engineered natural proteins. The formation of the
metallocarbenoid intermediate at the C45 heme facilitates not
1426
|
www.pnas.org/cgi/doi/10.1073/pnas.1915054117
Stenner et al.

data were fitted by using the following equation for reversible formation of
an unobserved tetrahedral intermediate, followed by irreversible removal of
dinitrogen to the stable metallocarbenoid species:
of LB containing the same concentrations of antibiotics (see above). The 1-L
cultures were grown in a shaking incubator at 37 °C and 180 rpm until an
OD600 between 0.6 and 0.8 was obtained (usually after 3 h), at which point
980 μL was extracted from both vessels, placed inside separate 1.5-mL screw-
top vials, and placed on ice. One milliliter of IPTG solution (1 M stock, 1 mM
final concentration at induction) was added to specific cultures to induce
protein expression; induced and noninduced cultures were left in the in-
cubator for an additional 3 h (37 °C and 180 rpm). After 3 h, 980 μL was
extracted from the vessels and transferred to separate 1.5-mL screw-top
vials. The samples were then degassed inside an anaerobic glove box
(Belle Technology) for 30 min before 10 μL of pyrrole (1 M stock in EtOH) was
added, and the vials were sealed and removed from the glove box. The vials
were cooled on ice before 25 μL of ethyl 2-bromo-2-diazoacetate (40 mM
stock in CH2Cl2, nitrogen flushed) was added via a gastight needle; the final
concentration of the reagents were 1 mM ethyl 2-bromo-2-diazoacetate and
10 mM pyrrole. After the reactions had been left stirring for 2 h, the samples
were quenched with 3 M HCl (30 μL) and extracted with 1 mL of ethyl acetate.
Analysis was conducted as reported in SI Appendix, Ring Expansion Assays.
kobs = klim½Sꢀ=K1 + ½Sꢀ. whereK1 = ðk−1 + k2Þ=k1.
[1]
Additional experiments using stopped-flow spectrophotometry were con-
ducted to study the degradation of the metallocarbenoid intermediates in
the presence of a suitable substrate. A solution containing a known con-
centration of ferrous C45/AP3.2/Rma-TDE (15 μM, 100 mM KCl, and 20 mM
CHES, pH 8.6, reduced with Na2S2O4) was placed in 1 syringe, and an 80:20%
ethanol:water solution containing 1 mM carbene precursor (either EDA,
tBuDA, or BnDA) and 6 mM styrene was placed in the 2nd syringe. Fifty
microliters from each syringe was simultaneously injected into a mixing
chamber, and the progression of the reaction was monitored spectroscopi-
cally, at 5 °C and 25 °C, over the course of 180 to 1,000 s to examine carbene
transfer activity. The progress of the reaction was monitored at 428 to 433 nm
and 417 nm, respectively. Final concentrations were 7.5 μM reduced C45/
AP3.2/Rma-TDE, 500 μM EDA/tBuDA/BnDA, and 3 mM styrene (40% ethanol).
Carbene Transfer Chemistry. Unless stated otherwise, all assays were con-
ducted under scrubbed nitrogen inside an anaerobic glove box ([O2] < 5 ppm;
Belle Technology). The assays were conducted inside 1.5-mL screw-top vials
sealed with a silicone–septum-containing cap. All assays were conducted in
CHES buffer (100 mM KCl and 20 mM CHES, pH 8.6), except for the assays
conducted with Mb(H64V,V68A), which were performed in KPi buffer
(100 mM potassium phosphate, pH 7).The final reaction volumes for all as-
says were 400 μL, unless otherwise stated. For a complete description of
individual assay conditions and product characterization by chiral HPLC and
LC-MS, please refer to the information provided in SI Appendix.
Supporting Information. Supporting methods, spectral and kinetic data, HPLC
and chiral HPLC chromatograms, LC-MS and MS data, HPLC calibrations, CD
spectra and thermal melts, a computational protein model mapped with AP3.2
mutations, and a scheme of NAD biosynthesis are provided in SI Appendix.
Data Availability. The data that support the findings of this study are available
from the corresponding author upon reasonable request.
ACKNOWLEDGMENTS. This work was supported at the University of Bristol
by Biotechnology and Biological Sciences Research Council (BBSRC) Grants
BBI014063/1, BB/R016445/1, BB/M025624/1; the Bristol Centre for Functional
Nanomaterials (Engineering and Physical Sciences Research Council [EPSRC]
Doctoral Training Centre Grant EP/G036780/1) through a studentship for
R.S.; and the SynBioCDT (EPSRC and BBSRC Centre for Doctoral Training in
Synthetic Biology Grant EP/L016494/1) through a studentship for J.W.S. We
thank Dr. Peter Wilson for assistance in collecting LC-MS and MS data; and
Dr. Steve Burston for helpful kinetic discussions.
Whole-Cell C45-Catalyzed Ring-Expansion Experiments. Overnight starter cul-
tures were prepared by adding 100 μL of carbenicillin (50 mg.mL−1) and
100 μL of chloramphenicol (50 mg.mL−1, C45 only) to 100 mL of Luria broth
(LB) before inoculating the medium with a C45-expressing E. coli glycerol
stock. Starter cultures were then incubated overnight at 37 °C and 180 rpm.
Fifty milliliters of the overnight starter culture was then used to inoculate 1 L
1. D. Röthlisberger et al., Kemp elimination catalysts by computational enzyme design.
Nature 453, 190–195 (2008).
2. C. Zeymer, R. Zschoche, D. Hilvert, Optimization of enzyme mechanism along the
evolutionary trajectory of a computationally designed (retro-)aldolase. J. Am. Chem.
Soc. 139, 12541–12549 (2017).
3. N. Preiswerk et al., Impact of scaffold rigidity on the design and evolution of an ar-
tificial Diels-Alderase. Proc. Natl. Acad. Sci. U.S.A. 111, 8013–8018 (2014).
4. N. S. Hong et al., The evolution of multiple active site configurations in a designed
enzyme. Nat. Commun. 9, 3900 (2018).
5. G. Caserta et al., Enhancement of peroxidase activity in artificial mimochrome VI
catalysts through rational design. Chembiochem 19, 1823–1826 (2018).
6. D. W. Watkins et al., Construction and in vivo assembly of a catalytically proficient and
hyperthermostable de novo enzyme. Nat. Commun. 8, 358 (2017).
7. A. E. Donnelly, G. S. Murphy, K. M. Digianantonio, M. H. Hecht, A de novo enzyme
catalyzes a life-sustaining reaction in Escherichia coli. Nat. Chem. Biol. 14, 253–255
(2018).
8. R. A. Snyder, S. E. Butch, A. J. Reig, W. F. DeGrado, E. I. Solomon, Molecular-level
insight into the differential oxidase and oxygenase reactivities of de Novo due ferri
proteins. J. Am. Chem. Soc. 137, 9302–9314 (2015).
9. M. L. Zastrow, A. F. A. Peacock, J. A. Stuckey, V. L. Pecoraro, Hydrolytic catalysis and
structural stabilization in a designed metalloprotein. Nat. Chem. 4, 118–123 (2011).
10. S. Studer et al., Evolution of a highly active and enantiospecific metalloenzyme from
short peptides. Science 362, 1285–1288 (2018).
11. R. Blomberg et al., Precision is essential for efficient catalysis in an evolved Kemp
eliminase. Nature 503, 418–421 (2013).
12. S. C. Patel, M. H. Hecht, Directed evolution of the peroxidase activity of a de novo-
designed protein. Protein Eng. Des. Sel. 25, 445–452 (2012).
20. P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Olefin cyclopropanation via
carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339,
307–310 (2013).
21. G. Sreenilayam, R. Fasan, Myoglobin-catalyzed intermolecular carbene N-H insertion
with arylamine substrates. Chem. Commun. (Camb.) 51, 1532–1534 (2015).
22. S. B. Kan, R. D. Lewis, K. Chen, F. H. Arnold, Directed evolution of cytochrome c for
carbon-silicon bond formation: Bringing silicon to life. Science 354, 1048–1051 (2016).
23. O. F. Brandenberg, R. Fasan, F. H. Arnold, Exploiting and engineering hemoproteins
for abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 47,
102–111 (2017).
24. K. Oohora et al., Catalytic cyclopropanation by myoglobin reconstituted with iron
porphycene: Acceleration of catalysis due to rapid formation of the carbene species. J.
Am. Chem. Soc. 139, 17265–17268 (2017).
25. L. Villarino et al., An artificial heme enzyme for cyclopropanation reactions. Angew.
Chem. Int. Ed. Engl. 57, 7785–7789 (2018).
26. M. Bordeaux, V. Tyagi, R. Fasan, Highly diastereoselective and enantioselective olefin
cyclopropanation using engineered myoglobin-based catalysts. Angew. Chem. Int. Ed.
Engl. 54, 1744–1748 (2015).
27. P. S. Coelho et al.,
A serine-substituted P450 catalyzes highly efficient carbene
transfer to olefins in vivo. Nat. Chem. Biol. 9, 485–487 (2013).
28. Z. J. Wang et al., Improved cyclopropanation activity of histidine-ligated cytochrome
P450 enables the enantioselective formal synthesis of levomilnacipran. Angew. Chem.
Int. Ed. Engl. 53, 6810–6813 (2014).
29. T. Heel, J. A. McIntosh, S. C. Dodani, J. T. Meyerowitz, F. H. Arnold, Non-natural olefin
cyclopropanation catalyzed by diverse cytochrome P450s and other hemoproteins.
Chembiochem 15, 2556–2562 (2014).
30. G. Sreenilayam, E. J. Moore, V. Steck, R. Fasan, Stereoselective olefin cyclopropanation
under aerobic conditions with an artificial enzyme incorporating an iron-chlorin e6
cofactor. ACS Catal. 7, 7629–7633 (2017).
31. H. Renata, Z. J. Wang, R. Z. Kitto, F. H. Arnold, P450-catalyzed asymmetric cyclo-
propanation of electron-deficient olefins under aerobic conditions. Catal. Sci. Technol.
4, 3640–3643 (2014).
13. V. Nanda, R. L. Koder, Designing artificial enzymes by intuition and computation. Nat.
Chem. 2, 15–24 (2010).
14. G. S. Murphy, J. B. Greisman, M. H. Hecht, De novo proteins with life-sustaining
functions are structurally dynamic. J. Mol. Biol. 428, 399–411 (2016).
15. T. A. Farid et al., Elementary tetrahelical protein design for diverse oxidoreductase
functions. Nat. Chem. Biol. 9, 826–833 (2013).
32. H. M. Key, P. Dydio, D. S. Clark, J. F. Hartwig, Abiological catalysis by artificial haem
proteins containing noble metals in place of iron. Nature 534, 534–537 (2016).
33. K. Oohora, Y. Kihira, E. Mizohata, T. Inoue, T. Hayashi, C(sp3)-H bond hydroxylation
catalyzed by myoglobin reconstituted with manganese porphycene. J. Am. Chem.
Soc. 135, 17282–17285 (2013).
16. R. L. Koder et al., Design and engineering of an O(2) transport protein. Nature 458,
305–309 (2009).
17. J. L. R. Anderson et al., Constructing a man-made c-type cytochrome maquette in vivo:
Electron transfer, oxygen transport and conversion to a photoactive light harvesting
maquette. Chem. Sci. (Camb.) 5, 507–514 (2014).
34. O. F. Brandenberg, K. Chen, F. H. Arnold, Directed evolution of a cytochrome P450
carbene transferase for selective functionalization of cyclic compounds. J. Am. Chem.
Soc. 141, 8989–8995 (2019).
18. T. L. Poulos, Heme enzyme structure and function. Chem. Rev. 114, 3919–3962 (2014).
19. E. Antonini, M. Brunori, Hemoglobin and Myoglobin in Their Reactions with Ligands
(North-Holland, Amsterdam, 1971), pp. 47–48.
Stenner et al.
PNAS
|
January 21, 2020
|
vol. 117
|
no. 3
|
1427

35. D. A. Vargas, A. Tinoco, V. Tyagi, R. Fasan, Myoglobin-catalyzed C-H functionalization
of unprotected indoles. Angew. Chem. Int. Ed. Engl. 57, 9911–9915 (2018).
36. M. J. Weissenborn et al., Enzyme-catalyzed carbonyl olefination by the E. coli protein
YfeX in the absence of phosphines. ChemCatChem 8, 1636–1640 (2016).
37. V. Tyagi, R. Fasan, Myoglobin-catalyzed olefination of aldehydes. Angew. Chem. Int.
Ed. Engl. 55, 2512–2516 (2016).
38. Z. J. Wang, N. E. Peck, H. Renata, F. H. Arnold, Cytochrome P450-catalyzed insertion of
carbenoids into N-H bonds. Chem. Sci. (Camb.) 5, 598–601 (2014).
39. J. A. McIntosh et al., Enantioselective intramolecular C-H amination catalyzed by
engineered cytochrome P450 enzymes in vitro and in vivo. Angew. Chem. Int. Ed.
Engl. 52, 9309–9312 (2013).
52. F. H. Arnold, Directed evolution: Bringing new chemistry to life. Angew. Chem. Int.
Ed. Engl. 57, 4143–4148 (2018).
53. J. E. Hall, J. V. Matlock, J. W. Ward, K. V. Gray, J. Clayden, Medium-ring nitrogen
heterocycles through migratory ring expansion of metalated ureas. Angew. Chem.
Int. Ed. Engl. 55, 11153–11157 (2016).
54. A. L. Crombie, J. L. Kane, Jr, K. M. Shea, R. L. Danheiser, Ring expansion-annulation
strategy for the synthesis of substituted azulenes and oligoazulenes. 2. Synthesis of
azulenyl halides, sulfonates, and azulenylmetal compounds and their application in
transition-metal-mediated coupling reactions. J. Org. Chem. 69, 8652–8667 (2004).
55. E. Büchner, T. Curtis, Synthesis of beta-keto esters from aldehydes and diazoacetic
acid. Ber. Dtsch. Chem. Ges. 18, 2371–2377 (1885).
40. I. Cho, Z.-J. Jia, F. H. Arnold, Site-selective enzymatic C‒H amidation for synthesis of
diverse lactams. Science 364, 575–578 (2019).
41. S. B. J. Kan, X. Huang, Y. Gumulya, K. Chen, F. H. Arnold, Genetically programmed
chiral organoborane synthesis. Nature 552, 132–136 (2017).
56. G. L. Ciamician, M. Dennstedt, Über die einwirkung des chloroforms auf die
kaliumverbindung pyrrols. Ber. Dtsch. Chem. Ges. 14, 1153–1163 (1881).
57. H. T. Bonge, B. Pintea, T. Hansen, Highly efficient formation of halodiazoacetates and
their use in stereoselective synthesis of halocyclopropanes. Org. Biomol. Chem. 6,
3670–3672 (2008).
42. C. C. Farwell, R. K. Zhang, J. A. McIntosh, T. K. Hyster, F. H. Arnold, Enantioselective
enzyme-catalyzed aziridination enabled by active-site evolution of
P450. ACS Cent. Sci. 1, 89–93 (2015).
a
cytochrome
58. J. Messens et al., The oxidase DsbA folds a protein with a nonconsecutive disulfide. J.
Biol. Chem. 282, 31302–31307 (2007).
43. Y. Wei, A. Tinoco, V. Steck, R. Fasan, Y. Zhang, Cyclopropanations via heme carbenes:
Basic mechanism and effects of carbene substituent, protein axial ligand, and por-
phyrin substitution. J. Am. Chem. Soc. 140, 1649–1662 (2018).
44. Y. Liu et al., Electronic configuration and ligand nature of five-coordinate iron por-
phyrin carbene complexes: An experimental study. J. Am. Chem. Soc. 139, 5023–5026
(2017).
59. E. Arslan, H. Schulz, R. Zufferey, P. Künzler, L. Thöny-Meyer, Overproduction of
the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in
Escherichia coli. Biochem. Biophys. Res. Commun. 251, 744–747 (1998).
60. B. San Francisco, M. C. Sutherland, R. G. Kranz, The CcmFH complex is the system I
holocytochrome c synthetase: Engineering cytochrome c maturation independent of
CcmABCDE. Mol. Microbiol. 91, 996–1008 (2014).
45. H. Renata et al., Identification of mechanism-based inactivation in P450-catalyzed
cyclopropanation facilitates engineering of improved enzymes. J. Am. Chem. Soc.
138, 12527–12533 (2016).
61. D. A. Mavridou, M. N. Clark, C. Choulat, S. J. Ferguson, J. M. Stevens, Probing heme
delivery processes in cytochrome c biogenesis system I. Biochemistry 52, 7262–7270
(2013).
46. R. D. Lewis et al., Catalytic iron-carbene intermediate revealed in a cytochrome c
carbene transferase. Proc. Natl. Acad. Sci. U.S.A. 115, 7308–7313 (2018).
47. R. L. Khade, Y. Zhang, Catalytic and biocatalytic iron porphyrin carbene formation:
Effects of binding mode, carbene substituent, porphyrin substituent, and protein
axial ligand. J. Am. Chem. Soc. 137, 7560–7563 (2015).
48. D. A. Sharon, D. Mallick, B. Wang, S. Shaik, Computation sheds insight into iron
porphyrin carbenes’ electronic structure, formation, and N-H insertion reactivity. J.
Am. Chem. Soc. 138, 9597–9610 (2016).
62. E. Henke, U. T. Bornscheuer, R. D. Schmid, J. Pleiss, A molecular mechanism of
enantiorecognition of tertiary alcohols by carboxylesterases. Chembiochem 4, 485–
493 (2003).
63. J. McLaren, D. T. C. Ngo, B. M. Olivera, Pyridine nucleotide metabolism in Escherichia
coli. 3. Biosynthesis from alternative precursors in vivo. J. Biol. Chem. 248, 5144–5149
(1973).
64. O. Kurnasov et al., NAD biosynthesis: Identification of the tryptophan to quinolinate
pathway in bacteria. Chem. Biol. 10, 1195–1204 (2003).
49. T. Hayashi et al., Capture and characterization of a reactive haem–carbenoid complex
in an artificial metalloenzyme. Nat. Catal. 1, 578–584 (2018).
50. M. Blaber, X.-J. Zhang, B. W. Matthews, Structural basis of amino acid alpha helix
propensity. Science 260, 1637–1640 (1993).
51. F. A. Tezcan, J. R. Winkler, H. B. Gray, Effects of ligation and folding on reduction
potentials of heme proteins. J. Am. Chem. Soc. 120, 13383–13388 (1998).
65. X. Wang et al., Engineering Escherichia coli nicotinic acid mononucleotide adenylyl-
transferase for fully active amidated NAD biosynthesis. Appl. Environ. Microbiol. 83,
e00692-17 (2017).
66. O. Bouvet, E. Bourdelier, J. Glodt, O. Clermont, E. Denamur, Diversity of the auxo-
trophic requirements in natural isolates of Escherichia coli. Microbiology 163, 891–
899 (2017).
1428
|
www.pnas.org/cgi/doi/10.1073/pnas.1915054117
Stenner et al.