Nitrene transfer catalyzed by a non-heme iron enzyme and enhanced by non-native small-molecule ligands

Article abstract of DOI:10.1021/jacs.9b11608

Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that Pseudomonas savastanoi ethylene-forming enzyme, a non-heme iron enzyme, can catalyze olefin aziridination and nitrene C-H insertion, and that these activities can be improved by directed evolution. The non-heme iron center allows for facile modification of the primary coordination sphere by addition of metal-coordinating molecules, enabling control over enzyme activity and selectivity using small molecules.

Full text of DOI:10.1021/jacs.9b11608

Subscriber access provided by University of Winnipeg Library
Communication
Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme
and Enhanced by Non-Native Small-Molecule Ligands
Nathaniel W. Goldberg, Anders M. Knight, Ruijie K. Zhang, and Frances H. Arnold
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 02 Dec 2019
Downloaded from pubs.acs.org on December 2, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and
Enhanced by Non-Native Small-Molecule Ligands
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†,§
‡,§
†,‖
†,‡,*
Nathaniel W. Goldberg , Anders M. Knight , Ruijie K. Zhang , Frances H. Arnold
†
‡
Division of Chemistry and Chemical Engineering and Division of Biology and Bioengineering, California Institute of
Technology, 1200 East California Boulevard, MC 210-41, Pasadena, California 91125, United States
Supporting Information Placeholder
ABSTRACT: Transition-metal catalysis is a powerful tool for the
O
O
O
O O
construction of chemical bonds. Here we show that Pseudomonas
savastanoi ethylene-forming enzyme (PsEFE), a non-heme iron
enzyme, can catalyze olefin aziridination and nitrene C–H
insertion, and that these activities can be improved by directed
evolution. The non-heme iron center allows for facile modification
of the primary coordination sphere by addition of metal-
coordinating molecules, enabling control over enzyme activity and
selectivity using small molecules.
L
Fe
O
S
S
S
Ph
N3
N
N
N
O
O
N
N
O
O
O
S
N3
NH
O
O
O
O
H
N
L =
-O
O-
-O
O-
-O
Over the last century, chemists have developed myriad synthetic
transition-metal catalysts to access new chemical transformations
and modes of reactivity. Nature has been developing catalysts for
far longer: over billions of years, she has evolved a rich repertoire
of proteins that perform most of the chemical reactions of life. But
nature’s inventions do not include many of the best inventions of
human chemists. Our efforts to merge abiological transition-metal
chemistry with nature’s vast toolbox of metalloproteins have
O
O
Figure 1. Small-molecule activation of a non-heme iron center for
nitrene transfer. Carboxylate-containing ligands α-ketoglutarate,
N-oxalylglycine, and acetate modulate the nitrene-transfer activity
of variants of P. savastanoi ethylene-forming enzyme.
To search for abiological catalytic promiscuity among natural
metalloproteins, we looked at α-ketoglutarate (αKG)-dependent
iron enzymes, a family of enzymes which features a conserved
metal-binding active site with iron coordinated by two histidines
and one aspartate or glutamate . In nature, these enzymes perform
similar chemistry to the heme-binding cytochrome P450 family, in
a high-valent iron-oxo intermediate performs C–H
hydroxylation, olefin epoxidation, or other oxidative
transformations . Though members of this enzyme family have
been reported to catalyze reactions beyond their native functions,
all the reactions reported proceed through the native iron-oxo
1
focused on heme-binding proteins , as the heme cofactor and its
analogues are well-studied in synthetic transition-metal chemistry.
However, heme-binding proteins represent only a small fraction of
the chemical diversity present in natural metalloproteins.
4
which
2
Metalloproteins comprise greater than 30% of all proteins and are
responsible for some of the most fundamental chemical reactions
in biology, including nitrogen fixation, photosynthesis, and DNA
synthesis. Natural metalloproteins bind a variety of metals in a wide
range of metal-binding sites, either coordinating the metal ion itself
or a more complex metal-containing cofactor. Nearly any
heteroatom-containing side chain can coordinate to a metal, in
addition to the peptide backbone, allowing myriad possible
5
6
mechanism . We hypothesized that non-heme iron enzymes might
also be able to catalyze abiological transformations similar to
heme-binding proteins through a non-natural mechanistic pathway.
3
We screened a set of seven purified α-ketoglutarate (αKG)-
dependent iron dioxygenases against the intermolecular
aziridination reaction of styrene 1 and p-toluenesulfonyl azide 2.
Aziridination and carbon–hydrogen (C–H) bond insertions of
nitrenes have been reported using engineered heme-binding
proteins and were subsequently proposed in a natural product
biosynthetic pathway . Chang and coworkers speculated the
existence of a transient iron-nitrene intermediate in their report of
the transformation of alkyl azides to nitriles by an αKG-dependent
iron dioxygenase, but this reaction still proceeds through the
coordination environments . Many coordination environments in
non-heme metalloenzymes have multiple open coordination sites at
the metal center, a key feature of numerous synthetic transition-
metal catalysts. Expanding new-to-nature catalysis to non-heme
metalloenzymes would open a new world of transition-metal
biocatalysis. In this work we show that a non-heme iron enzyme
can catalyze nitrene-transfer chemistry (Figure 1). This non-native
activity is enhanced by binding of non-native small-molecule
ligands and has been improved by directed evolution.
7
8
9
6b
canonical iron-oxo catalytic cycle . To date, no reported non-heme
iron enzyme, natural or engineered, has been reported to catalyze
productive nitrene transfer.
From the set of enzymes we tested, only Pseudomonas
savastanoi ethylene-forming enzyme (PsEFE, UniProt ID
P32021), formed aziridine 3 significantly above background
ACS Paragon Plus Environment

Journal of the American Chemical Society
Supplementary Table S2). Compared to other members of the
Table 1. Aziridination catalyzed by wild-type PsEFE
Page 2 of 5
(
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
αKG-dependent iron dioxygenase family PsEFE is mechanistically
and structurally distinct. While most enzymes of this family
catalyze the oxidation of a substrate, often C–H hydroxylation,
PsEFE natively catalyzes the fragmentation of the usual co-
substrate α‑ketoglutarate to ethylene, as well as the hydroxylation
O
O
S
N
N3
S
+
O
O
10
of L-arginine . Structurally, PsEFE possesses a hybrid fold,
1
2
3
combining elements of both type I and type II αKG-dependent iron
enzymes. It binds α-ketoglutarate in a strained conformation in an
unusually hydrophobic pocket, which is likely responsible for the
Deviation from standard conditions1
Relative activity
None
1.00
0.08
0.52
6.72
7.75
6.01
1
1
atypical catalytic activity .
No iron
No αKG
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As the iron-binding site in PsEFE is quite unlike that of the
heme-binding proteins that perform nitrene-transfer chemistry, we
sought to characterize the necessary components of the reaction.
Iron is required, with a single added equivalent of iron(II) sufficient
to fully restore catalytic activity of the wild-type apoenzyme.
PsEFE has three coordination sites filled by amino-acid side chains
(two histidines and one aspartate), leaving up to three additional
sites open for binding. In the native catalytic mechanism of PsEFE
and other members of its family, α-ketoglutarate occupies two of
these sites and is required for activity, as it is oxidatively
decarboxylated to succinate to generate the reactive iron-oxo
intermediate. PsEFE has been shown to catalyze arginine
hydroxylation with α-ketoadipate instead of α-ketoglutarate, but
with 500-fold lower activity. Other α-ketoacids were reported to
2
Acetate instead of αKG
3
N-oxalylglycine instead of αKG
Acetate instead of αKG, no ascorbic acid
1Standard conditions: Reactions were performed in MOPS
buffer (20 mM pH 7.0) with 5% ethanol co-solvent, with 20 μM
apoenzyme, 1 mM Fe(NH4) (SO )2, 1 mM αKG (as disodium
2 4
salt), 1 mM L-ascorbic acid, and 10 mM 1 and 2. Sodium salt.
2
3
Free acid.
We then sought to improve PsEFE for aziridination via directed
evolution, targeting active-site residues with site-saturation
mutagenesis and screening for enhanced activity. Details of our
engineering strategy are in the Supporting Information. Initial
screening for the first round was performed in the presence of
exogenous α-ketoglutarate, but for validation of this round and for
all subsequent evolution we screened with exogenous acetate.
Acetate was chosen as the preferred ligand because it enhanced the
activity of the wild-type enzyme significantly more than the native
α-ketoglutarate, it is biologically ubiquitous, and it is inexpensive.
Although α-ketoglutarate is the native ligand and is naturally
present at near-millimolar intracellular concentration in
1
2
give no activity . Nitrene transfer, however, does not proceed
through the native catalytic cycle and therefore does not require
αKG as a co-substrate; the αKG is now more a ligand and as such
could potentially be replaced by different small-molecule ligands.
Intrigued by the possibility of modulating enzyme activity by
changing the primary coordination sphere of the catalytic iron, we
tested PsEFE for aziridination with a set of α-ketoglutarate mimics
and related molecules as additives. We found that whereas addition
of a carboxylate is beneficial for activity (though not required), the
wild-type enzyme is significantly more active for aziridination with
added acetate or N-oxalylglycine (NOG, a general αKG-dependent
1
3
Escherichia coli , we reasoned that by supplementing the reaction
medium with acetate we could evolve PsEFE to be dependent on
acetate instead, despite screening under whole cell or lysate
conditions.
4
enzyme competitive inhibitor ) compared to α-ketoglutarate (Table
1
).
After two rounds of site-saturation mutagenesis and one round
of recombination, we found a variant with five mutations from the
wild type (T97M R171L R277H F314M C317M, PsEFE
MLHMM) which catalyzed the formation of 3 with 120 total
turnover number (TTN) and 88% enantiomeric excess (ee) favoring
the (R)-enantiomer (Figure 2A). Four of the five introduced
mutations are in the binding pocket of the native substrate arginine
and presumably are involved in substrate binding. The fifth
beneficial mutation is at Arg-277, the residue whose guanidino
group natively binds the distal carboxylate of α-ketoglutarate
(Figure 2B). The R277H mutation likely interferes with binding of
the native ligand α-ketoglutarate; as a result, PsEFE MLHMM
shows no significant increase in aziridination activity when α-
ketoglutarate is added, but an 11-fold increase when acetate is
added (Supplementary Table S4). Thus the evolved MLHMM
variant is highly activated by acetate but is no longer activated by
α-ketoglutarate at all, demonstrating the tunability of the ligand
dependence of PsEFE.
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
A
B
1
2
3
4
5
6
7
8
9
O
O
S
N
S
O
N3
+
O
1
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Directed evolution of PsEFE for aziridination. (A) Evolutionary lineage. Reactions were performed in triplicate anaerobically with
acetate and quantified by analytical HPLC-UV. Full experimental details are given in the Supporting Information. (B) Structural
representation of PsEFE with mutated sites highlighted in orange; metal-coordinating residues H189, D191, and H268 are represented in
sticks and Mn (the metal with which the protein was crystallized) is represented as a purple sphere (PDB ID: 6CBA).
We reasoned that variants of PsEFE generated by directed
evolution for aziridination could exhibit promiscuous activity for
additional nitrene-transfer reactions beyond aziridination.
Screening a panel of variants for the intramolecular C–H bond
insertion reaction of 2-ethylbenzenesulfonyl azide 4 to form sultam
Table 2. Nitrene C–H insertion catalyzed by PsEFE
O
O
O
O O
O
S
S
S
N3
NH2
NH
+
5
, we identified multiple variants that perform this reaction with
good TTN, excellent chemoselectivity, and moderate
enantioselectivity. Remarkably, PsEFE R171V F314M C317M
(PsEFE VMM) is significantly more active and more chemo- and
enantioselective with N-oxalylglycine added than with either
acetate or α-ketoglutarate, forming 5 with up to 730 TTN and
greater than 100:1 selectivity for insertion over reduction (Table 2).
This chemoselectivity is much higher than that of the heme proteins
previously reported to catalyze similar reactions . To probe the
specific effect of ligand binding on activity, we tested PsEFE
R171V R227H F314M C317M (PsEFE VHMM), a variant in
which αKG and NOG binding are disrupted by the R277H
mutation. Whereas PsEFE VHMM’s activity is still enhanced
nearly ten-fold with acetate, there is no significant difference
between its activity with no additive, αKG, or NOG added to the
reaction (Table 2). αKG or its analogues therefore appear to bind
within the native αKG binding site, activating the protein through
the primary metal coordination sphere.
4
5
6
PsEFE variant
Wild type
VHMM
VHMM
VHMM
VHMM
VMM
Additive
Acetate
None
TTN (5)
12
ee (%)
5/6
n.d.1
n.d.1
1.6
3.4
3.8
32
27
1
αKG
31
n.d.
8
a,b
Acetate
NOG
240
33
7.3
n.d.1
n.d.1
4.1
0.9
9.0
24
None
25
VMM
αKG
130
310
450
730
61
9.4
48
47
VMM
Acetate
NOG
VMM
VMM2
105
100
NOG
Aziridination with PsEFE is reasonably oxygen tolerant, with the
MLHMM variant maintaining 20% activity for aziridination when
performed in air. C–H insertion, however, does not proceed
detectably in aerobic conditions with any variant tested. These
observations suggest that formation of the putative iron-nitrene
intermediate is not significantly inhibited by oxygen, but that a
subsequent mechanistic step in the C–H insertion reaction is
Reactions were performed anaerobically in MOPS buffer
20 mM pH 7.0) with 2.5% ethanol co-solvent, 20 μM apoenzyme,
(
1
4 2 4 2
mM Fe(NH ) (SO ) , 1 mM additive, 1 mM L-ascorbic acid, and
10 mM 4 (maximum 500 TTN). Reactions were quantified by
1
analytical HPLC-UV. TTNs are shown for 5 only. Not determined
2
due to low conversion. 10 μM enzyme concentration (max. 1000
TTN).
8a
inhibited aerobically. Unlike nitrene transfer with heme proteins ,
PsEFE is highly expressed (>200 mg/L E. coli culture) and is
catalytically active for nitrene transfer in whole E. coli cells and in
cell lysate. We observe that in cell lysate the enzyme maintains high
activity with no external additive; the enzyme presumably retains a
ligand from the intracellular environment during lysis. Addition of
N-oxalylglycine nevertheless enhances C–H insertion yield and
chemoselectivity when PsEFE VMM is in cell lysate. In whole
an additional reductant is not required when reactions are
performed anaerobically (Supplementary Table S4).
ACS Paragon Plus Environment

Journal of the American Chemical Society
REFERENCES
Page 4 of 5
cells, however, there is no significant change upon addition of N-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
oxalylglycine (Supplementary Table S6). This is unsurprising as N-
oxalylglycine is not reported to be cell-permeable. Future
biochemical studies and further mutagenesis will likely enhance the
selectivity for ligand analogues and impart in vivo activation to
PsEFE variants.
(
1) Brandenberg, O. F.; Fasan, R.; Arnold, F. H. Exploiting and
engineering hemoproteins for abiological carbene and nitrene transfer
reactions. Curr. Opin. Biotechnol. 2017, 47, 102–111.
(2) Degtyarenko, K. Metalloproteins. In Encyclopedia of Genetics,
Genomics, Proteomics and Bioinformatics; Jorde, L.; Little, P.; Dunn, M.;
Subramaniam, S., Eds.; John Wiley & Sons, Ltd.: New York, 2005.
In conclusion, we have discovered a non-heme iron enzyme
capable of performing nitrene-transfer chemistry and enhanced that
activity via directed evolution. This is the first example of
enzymatic nitrene transfer catalyzed by a non-heme metalloprotein.
PsEFE features a metal center whose primary coordination sphere
can be altered by simple reaction additives, allowing for
modulation of catalytic activity and selectivity. We anticipate that
this biocatalytic system will lead to discovery of new
metalloenzymatic transformations not possible with previously
reported enzymes.
(
3) Holm, R. H.; Kennepohl, P.; Solomon, E. I. Structural and Functional
Aspects of Metal Sites in Biology. Chem. Rev. 1996, 96, 2239–2314.
4) Hausinger, R. P. Fe(II)/α-Ketoglutarate-Dependent Hydroxylases
and Related Enzymes. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 21–68.
5) Islam M. D.; Leissing, T. M.; Chowdhury, R.; Hopkinson, R. J.;
(
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Schofield, C. J. 2-Oxoglutarate-Dependent Oxygenases. Annu. Rev.
Biochem. 2018, 87, 585–620.
(6) (a) Matthews, M. L.; Chang, W.-c.; Layne, A. P.; Miles, L. A.; Krebs,
C.; Bollinger, J. M. Jr. Direct nitration and azidation of aliphatic carbons by
an iron-dependent halogenase. Nat. Chem. Biol. 2014, 10, 209–215. (b)
Davidson, M.; McNamee, M.; Fan, R.; Guo, Y.; Chang, W.-c. Repurposing
Nonheme Iron Hydroxylases To Enable Catalytic Nitrile Installation
through an Azido Group Assistance. J. Am. Chem. Soc. 2019, 141, 3419–
ASSOCIATED CONTENT
Supporting Information
3
423. (c) Neugebauer, M. E.; Sumida, K. H.; Pelton, J. G.; McMurry, J. L.;
Marchand, J. A.; Chang, M. C. Y. A family of radical halogenases for the
engineering of amino-acid-based products. Nat. Chem. Biol. 2019, 15,
1009–1016.
The Supporting Information is available free of charge on the ACS
Publications website.
Materials and experimental methods, compound characterization
data (PDF)
Full nucleotide and amino-acid sequences for all reported enzyme
variants and sequences of all oligonucleotides used for mutagenesis
(7) Farwell, C. C.; Zhang, R. K.; McIntosh, J. A.; Hyster, T. K.; Arnold,
F. H. Enantioselective Enzyme-Catalyzed Aziridination Enabled by Active-
Site Evolution of a Cytochrome P450. ACS Cent. Sci. 2015, 1, 89–93.
(8) (a) McIntosh, J. A.; Coelho, P. S.; Farwell, C. C.; Wang, Z. J.; Lewis,
J. C.; Brown, T. R.; Arnold. F. H. Enantioselective Intramolecular C–H
Amination Catalyzed by Engineered Cytochrome P450 Enzymes InꢀVitro
and InꢀVivo. Angew. Chem. Int. Ed. 2013, 52, 9309– 9312. (b) Singh, R.;
Bordeaux, M.; Fasan, R. P450-Catalyzed Intramolecular sp C–H
Amination with Arylsulfonyl Azide Substrates. ACS Catal. 2014, 4, 546–
(XLSX)
3
AUTHOR INFORMATION
Corresponding Author
5
52. (c) Prier, C. K.; Zhang, R. K.; Buller, A. R.; Brinkmann-Chen, S.;
Arnold, F. H. Enantioselective, intermolecular benzylic C–H amination
catalysed by an engineered iron-haem enzyme. Nat. Chem. 2017, 9, 629–
634.
*frances@cheme.caltech.edu
Present Address
‖Amyris Biotechnologies, Emeryville, California 94608, United
States
(9) Tsutsumi, H.; Katsuyama, Y.; Izumikawa, M.; Takagi, M.; Fujie, M.;
Satoh, N.; Shin-ya, K.; Ohnishi, Y. Unprecedented Cyclization Catalyzed
by a Cytochrome P450 in Benzastatin Biosynthesis. J. Am. Chem. Soc.
2
018, 140, 6631–6639.
10) Fukuda, H.; Ogawa, T.; Tazaki, M.; Nagahama, K.; Fujii, T.;
Author Contributions
(
§These authors contributed equally.
Tanase, S.; Morino, Y. Molecular cloning in Escherichia coli, expression,
and nucleotide sequence of the gene for the ethylene-forming enzyme of
Pseudomonas syringae pv. phaseolicola PK2. Biochem. Biophys. Res.
Commun. 1992, 188, 483–489.
(11) (a) Zhang Z., Smart T. J.; Choi, H.; Hardy, F.; Lohans, C. T.;
Abboud, M. I.; Richardson, M. S. W.; Paton, R. S.; McDonough, M. A.;
Schofield, C. J. Structural and stereoelectronic insights into oxygenase-
catalyzed formation of ethylene from 2-oxoglutarate. Proc. Natl. Acad. Sci.
U.S.A. 2017, 114, 4467–4672. (b) Martinez, S.; Fellner, M.; Ferr, C. Q.;
Ritchie, A.; Hu, J.; Hausinger, R. P. Structures and Mechanisms of the Non-
Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme:
Substrate Binding Creates a Twist. J. Am. Chem. Soc. 2017, 139, 11980–
11988.
(12) Martinez, S.; Hausinger, R. P. Biochemical and Spectroscopic
Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-dependent
Ethylene-Forming Enyzme from Pseudomonas syringae pv. phaseolicola
PK2. Biochemistry 2016, 55, 5989–5999.
(13) Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; Van Dien,
S. J.; Rabinowitz, J. D. Absolute metabolite concentrations and implied
enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 2009,
Notes
A provisional patent has been filed through the California Institute
of Technology based on the results presented here.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation
(NSF) Division of Molecular and Cellular Biosciences (grant
MCB-1513007). N. W. G., A. M. K., and R. K. Z. acknowledge
support from the NIH training grants NIH T32 GM07616 (N. W.
G.) and NIH T32 GM112592 (A. M. K., R. K. Z.) and NSF
Graduate Research Fellowship DGE-1144469 (A. M. K, R. K. Z.).
We thank Sabine Brinkmann-Chen for critical reading of the
manuscript and Noah P. Dunham, S. B. Jennifer Kan, and Benjamin
J. Levin for helpful discussions. We thank Professor Hans Renata
and Professor Harry Gray for generously sharing plasmids.
5
, 593–599.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
Insert Table of Contents artwork here
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
ACS Paragon Plus Environment