An artificial metalloenzyme with the kinetics of native enzymes

Article abstract of DOI:10.1126/science.aah4427

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C-H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours^-1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C-H bonds and intermolecular carbene insertions into C-H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.

Full text of DOI:10.1126/science.aah4427

RESEARCH
| REPORTS
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Energy Sciences, Materials Sciences and Engineering Division of
the U.S. Department of Energy under contract DE-AC02-
05CH11231. A.B.S. was funded by Applied Materials, Inc., and
Entegris, Inc., under the I-RiCE program. J.P.L. and J.B. were
supported in part by the Office of Naval Research BRC program.
J.P.L. acknowledges a Berkeley Fellowship for Graduate Studies
and the NSF Graduate Fellowship Program. Q.W. and M.J.K. were
supported by the NRI SWAN Center and Chinese Academy of
Sciences President’s International Fellowship Initiative
(2015VTA031). G.P. and H.-S.P.W. were supported in part by the
SONIC Research Center, one of six centers supported by the
STARnet phase of the Focus Center Research Program (FCRP) a
Semiconductor Research Corporation program sponsored by
MARCO and DARPA. A.J., H.-S.P.W., and J.B. acknowledge the NSF
Center for Energy Efficient Electronics Science (E3S). A.J.
acknowledges support from Samsung. The authors acknowledge
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are reported in the main text and supplementary materials.
The effect of MoS2 thickness on the device
characteristics was systematically explored. At the
scaling limit of the gate length, the semiconductor
channel thickness must also be scaled down ag-
gressively, as described earlier. The electrostatic
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decreased with increasing distance from the ZrO2-
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ness was qualitatively consistent with the TCAD
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The unwanted variations in device performance
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and fig. S10), and the need for low Off state cur-
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justify the need for layered semiconductors like
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SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/354/6308/99/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S10
Table S1
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ACKNOWLEDGMENTS
S.B.D. and A.J. were supported by the Electronics Materials
program funded by the Director, Office of Science, Office of Basic
30 June 2016; accepted 7 September 2016
10.1126/science.aah4698
BIOCATALYSIS
TMDs offer the ultimate scaling of thickness
with atomic-level control, and the 1D2D-FET
structure enables the study of their physics and
electrostatics at short channel lengths by using
the natural dimensions of a SWCNT, removing
the need for any lithography or patterning pro-
cesses that are challenging at these scale lengths.
However, large-scale processing and manufac-
turing of TMD devices down to such small gate
lengths are existing challenges requiring future
innovations. For instance, research on develop-
ing process-stable, low-resistance ohmic contacts
to TMDs, and scaling of the gate dielectric by
using high-k 2D insulators is essential to further
enhance device performance. Wafer-scale growth
of high-quality films (30) is another challenge
toward achieving very-large-scale integration of
TMDs in integrated circuits. Finally, fabrication
of electrodes at such small scale lengths over large
areas requires considerable advances in litho-
graphic techniques. Nevertheless, the work here
provides new insight into the ultimate scaling of
gate lengths for a FET by surpassing the 5-nm
limit (3–7) often associated with Si technology.
An artificial metalloenzyme with the
kinetics of native enzymes
P. Dydio,1,2* H. M. Key,1,2* A. Nazarenko,1 J. Y.-E. Rha,1 V. Seyedkazemi,1
D. S. Clark,3,4 J. F. Hartwig1,2
†
Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities.
Although artificial metalloenzymes have been developed that catalyze abiological
transformations with high stereoselectivity, the activities of these artificial enzymes are
much lower than those of natural enzymes. Here, we report a reconstituted artificial
metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to
those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium
in place of iron catalyze insertions of carbenes into C–H bonds with up to 98% enantiomeric
excess, 35,000 turnovers, and 2550 hours−1 turnover frequency. This activity leads to
intramolecular carbene insertions into unactivated C–H bonds and intermolecular carbene
insertions into C–H bonds. These results lift the restrictions on merging chemical
catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes
for abiological reactions.
he catalytic activity of a metalloenzyme is
determined by both the primary coordina-
tion sphere of the metal and the surrounding
protein scaffold. In some cases, laboratory
evolution has been used to develop variants
actions for which there is no known enzyme
(abiological transformations) (3, 4).
Although the reactivity of these artificial sys-
tems is new for an enzyme, the rates of these
reactions have been much slower and the
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T
of native metalloenzymes for selective reactions
of unnatural substrates (1, 2). Yet with few ex-
ceptions (3), the classes of reactions that such
enzymes undergo are limited to those of bio-
logical transformations. To combine the favorable
qualities of enzymes with the diverse reactivity of
synthetic transition-metal catalysts, abiological
transition-metal centers or cofactors have been
incorporated into native proteins. The resulting
artificial metalloenzymes catalyze classes of re-
1Department of Chemistry, University of California, Berkeley,
CA 94720, USA. 2Chemical Sciences Division, Lawrence
Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA
94720, USA. 3Department of Chemical and Biomolecular
Engineering, University of California, Berkeley, CA 94720,
USA. 4Molecular Biophysics and Integrated Bioimaging
Division, Lawrence Berkeley National Laboratory, 1 Cyclotron
Road, Berkeley, CA 94720, USA.
*These authors contributed equally to this work. †Corresponding
author. Email: jhartwig@berkeley.edu
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turnover number (TON) much lower than those
of reactions catalyzed by free metal complexes in
organic solvents and lower than those typical of
natural enzymes (4, 5). In addition to lacking
high activity, these enzymes lack practical char-
acteristics of enzymes for synthetic applications,
such as suitability for preparative-scale reactions
and potential to be recovered and reused (6). One
reason that artificial metalloenzymes react more
slowly than native enzymes is the absence of a
defined binding site for the substrate. Natural
enzymes generally bind their substrates with high
affinity and in a conformation that leads to ex-
tremely fast rates and high selectivity (7). If the
artificial metalloenzyme is generated by incor-
porating a full metal-ligand complex into the
substrate-binding site of a natural enzyme or
protein, the space remaining to bind a reactant
for a catalytic process is limited, and the inter-
actions by which the protein binds the reactant
are compromised (4, 8). Recently, we reported
artificial metalloenzymes generated by the formal
replacement of an abiological metal for the na-
tural iron in myoglobin that catalyzes abiological
reactions (9), but the activity of the resulting en-
zymes was far from that needed for synthetic
applications (6). Here, we show that artificial
metalloenzymes created by substituting an iridium-
methyl unit for the iron in cytochrome P450
enzymes (10) and modified by means of labora-
tory evolution catalyze abiological reactions with
activities that are comparable with those of native
enzymes (5).
P450 enzymes constitute a superfamily of heme-
binding monooxygenases that are involved in
various biosynthetic pathways, catalyzing re-
actions that encompass chemo-, regio-, stereo-,
and site-selective C–H hydroxylations of complex
natural products (11). We hypothesized that ar-
tificial metalloenzymes that are created with
P450s from thermophiles, such as CYP119 (12)
from Sulfolobus solfataricus, could improve the
thermal stability of the resulting artificial metal-
loenzyme and create the potential to conduct re-
actions at elevated temperatures. Studies on stability
revealed that the Ir(Me)-PIX protein formed from
CYP119 did have a much higher melting temper-
ature (Tm = 69°C) than those formed from the
more commonly used P450-BM3 (45°C) or P450-
CAM (40°C). Therefore, this protein was used for
our studies on catalytic reactions.
5 mM 1 and 0.1 mole % (mol %) catalyst. (Single-
letter abbreviations for the amino acid residues
are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F,
Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met;
N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V,
Val; W, Trp; and Y, Tyr. In the mutants, amino
acids were substituted at specific locations; for
example, C317G indicates that cysteine at posi-
tion 317 was replaced by glycine.) To identify
mutants that form 2 with higher rates and en-
antioselectivity, we used a directed evolution
strategy targeting the residues close to the active
site (L69, A209, T213, and V254) (Fig. 2). To retain
the hydrophobicity of the active site, we intro-
duced only hydrophobic and uncharged resi-
dues (V, A, G, F, Y, S, and T) through site-directed
mutagenesis within a library of 24 double mutants
of CYP119. The mutant C317G, T213G formed 2with
68% ee and 80-fold higher activity (TOF = 9.3 min−1)
than did that of the single mutant C317G under the
same reaction conditions, described previously.
Two additional rounds of evolution, in which
~150 additional variants were analyzed, led to
the quadruple mutant C317G, T213G, L69V, V254L
(hereafter referred to as CYP119-Max), which formed
2 with 94% ee and with an initial TOF of 43 min−1.
This rate is more than 180 times faster than that
of the WT variant.
substrate 1 for the WT enzyme is weak, as re-
vealed by a high KM (> 5 mM). The single mutant
C317G, which lacks a sidechain at this position
that could act as an axial ligand, exhibits higher
substrate affinity (KM = 3.1 mM), catalytic activity
(kcat = 0.22 min−1), and therefore overall enzyme
efficiency (kcat/KM = 0.071 min−1 mM−1) than that
of the WT enzyme. The double mutant T213G,
C317G of Ir(Me)-CYP119 reacts with Michaelis-
Menten parameters (kcat = 4.8 min−1 and KM
=
0.40 mM, kcat/KM = 12 min−1 mM−1) that are far
more favorable than those of the single mutant
C317G. The incorporation of two additional muta-
tions (L69V, V254L) led to further improvements
of both kcat and KM, creating an enzyme (CYP119-
Max) with an efficiency that is greater than
4000 times (kcat = 45.8 min−1, KM = 0.17 mM, and
kcat/KM = 269 min−1 mM−1) that of the WT sys-
tem. These kinetic parameters mark a vast im-
provement over those of the variant of myoglobin
Ir(Me)-PIX-mOCR-Myo H93A, H64V (kcat = 0.73 min−1,
KM = 1.1 mM, and kcat/KM = 0.66 min−1mM−1)
(Fig. 2) (15). Furthermore, the rates of reactions
catalyzed by CYP119-Max at concentrations below
KM are more than 20 times higher than those
catalyzed by the free iridium-porphyrin in the
presence of the same substrate concentration
(TOF = 21 min−1 versus TOF = 0.93 min−1 at
0.15 mM 1, respectively), even though the free
cofactor lacks any steric encumbrance near the
metal site necessary to enable selective catalysis.
These results show the value of conducting this
iridium-catalyzed reaction within the enzyme
active site to control selectivity and increase
the reaction rate simultaneously.
Kinetic studies provided insight into the origin
of the differences in the enzymatic activity be-
tween the various mutants of Ir(Me)-CYP119. In
particular, we determined the standard Michaelis-
Menten kinetic parameters (kcat and KM) for the
mutants at each stage of the evolution. Using
these parameters, we determined the catalytic ef-
ficiency of each enzyme, which is defined as kcat
/
The kinetic parameters of reactions catalyzed
by the Ir(Me)-PIX CYP119-Max enzyme are com-
parable with those of native reactions catalyzed
by the natural enzymes involved in intermediate
KM (14) and considered to be one of the most
relevant parameters for comparing engineered
enzymes to natural enzymes (5). The affinity of
By studying the model reaction to convert
diazoester 1 into dihydrobenzofuran 2, which
does not occur in the presence of natural Fe-PIX
enzymes, we found that the activity and selec-
tivity of Ir(Me)-PIX CYP119 enzymes are readily
evolved through molecular evolution of the nat-
ural substrate-binding site of the CYP119 scaffold
(Figs. 1 and 2). The wild type (WT) Ir(Me)-CYP119
enzyme and its variant C317G [bearing a mutation
that introduces space to accommodate the axial
ligand of the Ir(Me)-PIX cofactor] (13) catalyze
the intramolecular carbene insertion into a C–H
bond to form 2, although with low rates [turn-
over frequency (TOF) = 0.23 and 0.13 min−1
respectively] and enantioselectivities (ee = 0 and
14%, respectively) for reactions conducted with
,
Fig. 1. Structure of WT Fe-CYP119. Image was prepared in Chimera from Protein Data Bank 1IO7.
(Left) Complete structure of Fe-CYP119. (Right) Active-site residues modified during directed evolution of
the protein scaffold to increase activity and selectivity for carbene insertions into C–H bonds.
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and secondary metabolism (5), such as many
cytochromes P450 (16). The binding affinity of
Ir(Me)-CYP119-Max for the abiological substrate
1 is even higher than the affinity of P450s for
their native substrates (compare KM = 0.17 mM
for CYP119-Max with KM = 0.298 mM for P450-
BM3 and its native substrate lauric acid) (17) and
similar to the median KM value for natural en-
zymes of this class (0.13 mM) (5). In addition,
the kcat of 45.8 min−1 for this enzyme is within
an order of magnitude of the median kcat of nat-
ural enzymes responsible for the production of
biosynthetic intermediates (312 min−1) and sec-
ondary metabolites (150 min−1) (5).
reactive substrates (Fig. 3). Directed evolution
targeting high stereoselectivity led to mutants
that form products 3 to 7 in up to 98% ee, in
reactions conducted with a fixed catalyst loading
(0.17 mol %). The reactions to form products 3
to 5 show that the enzyme reacts as selectively
with substrates containing substituents on the
aryl ring as it does for unsubstituted 2. Such re-
activity is relevant to contemporary synthetic
challenges. For example, compound (S)-5 is an
intermediate in the synthesis of BRL 37959—a
potent analgesic—and was prepared previously
by means of kinetic resolution (18). This product
was formed by variant 69Y-152W-213G in 94% ee.
Product 6 results from carbene insertion into a
secondary C–H bond, and product 7 results from
carbene insertion into a sterically hindered,
secondary C–H bond. Directed evolution fur-
nished a mutant capable of forming 7 with high
enantio- and diastereoselectivity (dr) favoring
the cis isomer [90% ee, 12:1 dr (cis:trans)]. The
previously reported Ir(Me)-PIX enzymes based on
myoglobin produced this product in only trace
amounts, and the free Ir(Me)-PIX cofactor formed
predominantly the trans isomer (3:1 dr, trans:cis).
This reversal of diastereoselectivity from that of
the free Ir(Me)-PIX cofactor to that of the artificial
metalloenzyme highlights the ability of strong
substrate-enzyme interactions to override the in-
herent selectivity of a metal cofactor or substrate.
Ir(Me)-CYP119-Max also catalyzes the insertion
of carbenes into fully unactivated C–H bonds. Al-
though the structure of substrate 7 in Fig. 4 appears
similar to that of substrate 1, the primary C–H
bonds in 7 are stronger and less reactive than those
in 1, which are located alpha to an oxygen atom (19).
The potential to evolve proteins having advan-
tageous enzyme-substrate interactions should also
create the possibility to catalyze the insertion of
carbenes into the C–H bonds of diverse and less
Fig. 2. Directed evolution of Ir(Me)-PIX CYP119 for enantioselective inser-
tions of carbenes into C–H bonds. (A) Model reaction converting diazoester
1 to dihydrobenzofuran 2. (B) Enantioselectivity and yields for the formation of
2 catalyzed by evolved variants of CYP119 (0.17% catalyst loading, 10 mM
substrate). (C) Kinetic parameters describing the formation of 2 by variants of
CYP119 (0.1 mol % catalyst loading, 5 mM substrate). For free Ir(Me)-PIX, k1
(the first-order kinetic constant) is listed instead of kcat/KM (16). (Inset) De-
pendence of TOF on the initial concentration of 1 for reactions using 0.005 mM
catalyst. (D and E) Comparison of KM and kcat values for CYP119-Max with those
of natural enzymes involved in the metabolism of intermediate and secondary
metabolites (5); for comparison, the kinetic parameters for Ir(Me)-PIX mOCR-
myoglobin (H93A and H64V) catalyzing the same transformation are shown.
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In fact, there are no metal catalysts of any type
reported to form indanes via carbene insertion
into an unactivated C–H bond with synthetically
useful enantioselectivities. The synthesis of such
chiral moieties was achieved only in a diaster-
eoselective fashion, when a stoichiometric amount
of a chiral auxiliary was built into the substrate
(20). In contrast, Ir(Me)-CYP119-Max catalyzed the
formation of 8 in 90% ee, with no need for the
auxiliary. To observe substantial amounts of this
product (TON = 31), the reaction was conducted
at 40°C; the enantioselectivity of the product at
this temperature was the same as that of the small
quantity of product formed at room temperature.
In addition to catalyzing intramolecular reac-
tions with unactivated C–H bonds, Ir(Me)-CYP119-
Max catalyzes intermolecular carbene insertion
into a C–H bond. This reaction is challenging be-
cause the metal-carbene intermediate can undergo
competitive diazo coupling or insert the carbene
unit into the O–H bond of water (21, 22). In fact,
the model reaction between phthalan (10) and
ethyl diazoacetate (EDA) forms alkene and alcohol
as the dominant products when catalyzed by the
Fig. 3. Selective variants of Ir(Me)-PIX CYP119.These variants catalyze enantioselective intra- and intermolecular C–H carbene insertion reactions of activated
and unactivated C–H bonds. (A to D) Intramolecular C–H carbene insertion reactions. Reactions were conducted at room temperature unless otherwise noted.
(E) An intermolecular C–H carbene insertion reaction. Conditions: 10 (10 mmol) and EDA (100 mmol). EDA was added as a 50% solution in N,N′-dimethylformamide
over 1 hour by use of a syringe pump.
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Fig. 4. Productivity of Ir(Me)-PIX CYP119. Intramolecular C–H carbene insertion reactions of substrate 1 catalyzed by Ir(Me)-PIX CYP119-Max under synthetically
relevant reaction conditions.
free Ir(Me)-PIX cofactor; only trace amounts of
carbene insertion product 11 were formed. In
sharp contrast, the same reaction catalyzed by
the mutant Ir(Me)-PIX CYP119-Max-A152F oc-
curred to form 11 in 55% yield with 330 TON
and 68% ee. Dimerization of the carbene when
catalyzed by Ir(Me)-CYP119-Max is limited, pre-
sumably because of selective binding and pre-
organization of the substrate; the selectivity for
formation of the product from C–H insertion
11 over the alkene side product 12 was 70-fold
higher when catalyzed by Ir(Me)-PIX CYP119-
Max-A152F than when catalyzed by the free
cofactor.
formation of 2, while retaining 64% of the ac-
tivity (fig. S11).
15. This variant of myoglobin was used for comparison
because this variant forms 2 with both high yield and
high enantioselectivity [relative to other mutants of
Ir(Me)-PIX Myo].
16. J. Romeo, Ed., Secondary Metabolism in Model Systems
(Elsevier, 2004).
17. M. A. Noble et al., Biochem. J. 339, 371–379
(1999).
18. P. Bongen, J. Pietruszka, R. C. Simon, Chemistry 18,
11063–11070 (2012).
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2036–2039 (2012).
20. B. Hong et al., J. Am. Chem. Soc. 137, 11946–11949
(2015).
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Commun. 6, 7789 (2015).
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(2016).
Enzymes containing abiological transition-metal
active sites that exhibit the kinetics, selectivity, and
evolutionary potential of natural enzymes have
been a major goal of catalyst design. Here, we
show that artificial metalloenzymes catalyzing
abiological processes can possess the fundamental
characteristics of natural enzymes: fast kinetics,
high productivity, and high selectivity under the
same reaction conditions. Taken together, our re-
sults show that the kinetics of artificial metal-
loenzymes need not limit the merging of chemical
and biocatalysis.
For the ultimate goal of applying artificial
metalloenzymes to the synthesis of organic mol-
ecules for fine chemicals, the reactions conducted
by such catalytic systems should occur on prepa-
rative scales with high substrate concentrations,
and the enzyme should react with high TONs and
be amenable to attachment to a solid support for
recycling. We found that a series of reactions con-
taining between 40 mg and 1 g of substrate
1 catalyzed by Ir(Me)-CYP-Max occurred with
yields and enantioselectivities that were sim-
ilar to each other (91 to 94% ee), showing that
the outcome of the reaction is independent of
the scale (Fig. 4). Moreover, with 200 mM substrate,
reactions catalyzed by Ir(Me)-CYP-Max (0.0025 mM)
formed product 2 with up to 35,000 TON without
loss of enantioselectivity (93% ee) (Fig. 4). Thus,
this artificial metalloenzyme operates with high
productivity under conditions suitable for pre-
parative scales. Last, Ir(Me)-CYP-Max supported
on CNBr-activated sepharose catalyzed the con-
version of 1 to 2 via carbene insertion into a C–
H bond in 52% yield and 83% ee. This supported
catalyst was used, recovered, and recycled four
times without loss of the enantioselectivity for
ACKNOWLEDGMENTS
REFERENCES AND NOTES
This work was supported by the Director, Office of Science, of
the U.S. Department of Energy under contract DE-AC02-
05CH11231; by the NSF (graduate research fellowship to H.M.K.),
and the Netherlands Organization for Scientific Research (the
Rubicon postdoctoral fellowship 680-50-1306 to P.D.). We thank
the QB3 MacroLab facility (competent cells), the University of
California, Berkeley DNA Sequencing Facility (plasmid sequencing),
and T. Iavarone and the QB3 Mass Spectrometry Facility
[nano–electrospray ionization (NS-ESI) mass spectrometry
data collection, supported by NIH grant 1S10OD020062-01]
for native nanoESI-MS data and analysis. All data are provided
in the supplementary materials. J.F.H., H.M.K., P.F.D., and
D.S.C. are inventors on U.S. patent application numbers
62/241,487 and 62/384,011, submitted by the Lawrence
Berkeley National Laboratory, that covers preparation and
application of artificial metalloenzymes containing noble
metal-porphyrins.
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SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/354/6308/102/suppl/DC1
Materials and Methods
Figs. S1 to S12
Tables S1 to S7
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28 June 2016; accepted 12 September 2016
10.1126/science.aah4427
106 7 OCTOBER 2016 • VOL 354 ISSUE 6308
sciencemag.org SCIENCE

An artificial metalloenzyme with the kinetics of native enzymes
P. Dydio, H. M. Key, A. Nazarenko, J. Y.-E. Rha, V. Seyedkazemi,
D. S. Clark and J. F. Hartwig (October 6, 2016)
Science 354 (6308), 102-106. [doi: 10.1126/science.aah4427]
Editor's Summary
Something like the real thing
Artificial metalloenzymes ideally combine the favorable properties of natural enzymes with the
high efficiency of synthetic catalysts. Inserting new metal groups into existing native proteins, however,
often leads to poorer overall catalytic efficiency. To break through this limitation, Dydio et al. replaced
the iron in the heme group of cytochrome P450 with iridium and subjected it to directed evolution. The
enzyme catalyzed a range of reactions with kinetics similar to those of the native enzyme. It was also
able to functionalize fully unactivated C-H bonds, a reaction that previously has only been mediated by
synthetic catalysts. Moreover, the artificial enzyme was stable across temperatures and scales that are
used industrially.
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