A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

Article abstract of DOI:10.1002/anie.201901782

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high-throughput mass-spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click-assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450_BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.

Full text of DOI:10.1002/anie.201901782

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: A high-throughput mass spectrometric enzyme activity assay
enabling the discovery of cytochrome P450 biocatalysts
Authors: Tristan de Rond, Jian Gao, Amin Zargar, Markus de Raad,
Jack Cunha, Trent R Northen, and Jay D Keasling
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901782
Angew. Chem. 10.1002/ange.201901782
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http://dx.doi.org/10.1002/ange.201901782

10.1002/anie.201901782
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COMMUNICATION
A high-throughput mass spectrometric enzyme activity assay
enabling the discovery of cytochrome P450 biocatalysts
Tristan de Rond*, Jian Gao, Amin Zargar, Markus de Raad, Jack Cunha, Trent R. Northen, and Jay D.
Keasling*
Abstract: Assaying for enzymatic activity is a persistent bottleneck in
biocatalyst and drug development. Existing high-throughput assays for
enzyme activity tend to be applicable only to a narrow range of
change their substrates’ masses. However, due to ionization suppression
and isobaric ions, MS analysis is limited to low-complexity samples.
This poses a significant disadvantage when screening large enzyme
libraries for a desired catalytic activity, or evaluating enzymatic activity
exhibited by clinical samples, both of which are typically conducted in
biochemical
transformations,
whereas
universal
enzyme
characterization methods usually require chromatography to determine
substrate turnover, greatly diminishing throughput. We present an
enzyme activity assay which allows for the high-throughput mass-
spectrometric detection of enzyme activity in complex matrices without
the need for a chromatographic step. We demonstrate that this
technology, which we call “Probing Enzymes with ‘Click’–Assisted
NIMS” (PECAN), can detect the activity of medically and
complex biological matrices. Coupling MS analysis to
a
chromatographic step, such as in liquid chromatography-MS (LC-MS)
or gas chromatography-MS (GC-MS), alleviates ionization and spectral
complexity issues by physically separating analytes, but significantly
decreases throughput.
One promising platform for high-throughput enzyme activity
biocatalytically significant cytochrome P450s in cell lysate, microsomes, determination is Nanostructure-Initiator Mass Spectrometry (NIMS)[8–
and bacterial cells. Using our approach, we successfully screened a
cytochrome P450BM3 mutant library for the ability to catalyze the
oxidation of the sesquiterpene valencene.
10]. NIMS involves laser desorption of analytes from a fluorophilic
surface. Fortuitously, this surface has high affinity for
perfluoroalkylated analytes through non-covalent fluorous interactions,
enabling the substitution of lengthy chromatographic separations with
an in-situ washing step. However, many enzymes may be unable to
accommodate perfluoroalkylated substrates in their active sites, which
may be why NIMS-based enzyme assays have thus far been applied only
Enzyme screening campaigns are frequently the bottleneck of drug,
biomarker and biocatalyst discovery programs, making high-throughput
enzyme activity assays essential to accelerating research in these areas[1–
4]. Such screens often rely on fluorogenic or chromogenic enzyme
substrate analogs, coupled assays, or biosensors, which – while high-
throughput – are unsuitable for monitoring many important enzymatic
to carbohydrate-acting enzymes and acetyltransferases[8,9,11–13]
In this work, we carry out an enzymatic reaction on a substrate
analog (“probe”), after which we couple the probe to
.
a
perfluoroalkylated affinity tag using copper-catalyzed “click”
chemistry[14] (Fig. 1). This approach, which we call “Probing Enzymes
with ‘Click’–Assisted NIMS” (PECAN), requires only the incorporation
of a relatively small “clickable” handle into the probe. When combined
with acoustic sample deposition and MS Imaging[15], this permits high–
throughput characterization of enzyme activity in complex biological
matrices.
transformations[5–7]
.
Mass spectrometry (MS) is a promising technology for the analysis
of enzyme activity, given that the majority of enzymatic reactions
[*] Tristan de Rond, Jay Keasling
College of Chemistry, University of California, Berkeley
Berkeley, CA 94270 (USA)
E-mail: tristan.de.rond@berkeley.edu, keasling@berkeley.edu
Tristan de Rond, Amin Zargar, Jack Cunha, Trent Northen, Jay Keasling
Joint Bioenergy Institute (JBEI)
Lawrence Berkeley National Laboratory
Emeryville, CA 94608 (USA)
Tristan de Rond
We apply PECAN to cytochrome P450s (“P450s”) which are an
important class of enzymes possessing the remarkable capacity to effect
regio– and stereospecific C–H bond activation reactions. P450s play
important roles in the biosynthesis of natural products[16] and steroids[17]
,
and are central to human xenobiotic metabolism[18]. Their ability to
catalyze reactions that are outside of the reach of traditional synthetic
organic chemistry has made P450s valuable biocatalysts. Consequently,
much research has been directed towards identifying P450s with the
appropriate substrate–, regio– and stereospecificity for industrial
Current Affiliation: Scripps Institution of Oceanography
University of California, San Diego
La Jolla, CA 92037 (USA)
Jian Gao, Markus de Raad, Trent Northen
Department of Energy Joint Genome Institute (DOE JGI)
Lawrence Berkeley National Laboratory
Markus de Raad, Trent Northen
Environmental Genomics and Systems Biology
Lawrence Berkeley National Laboratory
Jay Keasling
Biological Systems and Engineering Division
Lawrence Berkeley National Laboratory
Jay Keasling
biotransformations[19–21]
developed to facilitate these studies include chromogenic[22–24]
.
High-throughput P450 activity assays
,
fluorometric[25], luminogenic[26] and coupled assays[27], as well as MS-
based approaches harnessing the high mass resolution of FT-ICR MS to
distinguish analytes from matrix ions[28] or solid-phase extraction to
enrich for analytes prior to MS analysis[29]
.
To benchmark the PECAN technology, we tested its ability to detect
the activity of cytochrome P450BM3, a model P450 widely employed in
biocatalysis[30]. P450BM3 is known to catalyze the hydroxylation of 1a to
form 2a with a 34% e.e. of R-2a[31] (Fig. 2a), We designed an analog of
1a harboring an azide, 1b, to act as PECAN probe (Fig. 2b). We fed 1b
(0.5 mM in 1% v/v DMSO) to lysates of E. coli expressing P450BM3 or
Green Fluorescent Protein (GFP, serving as a negative control), along
with a cofactor regeneration system (10 mM glucose 6-phosphate, 100
Center for Biosustainability,
Danish Technical University, Lyngby, Denmark
Jay Keasling
Center for Synthetic Biochemistry, Institute for Synthetic Biology
Shenzhen Institutes of Advanced Technology, Shenzhen, China
Supporting information for this article is given via a link at the end of the
document
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Analyte-specific
tagging using
“click” chemistry
Cell lysate or other Add clickable
Enzyme
activity
complex mixture
substrate
(“probe”)
Cu+
m/z
m/z
Acoustic
In situ cleanup:
Nanostructure-
Change in
printing onto
NIMS surface
fluorous surface retains
only tagged probe
Initiator Mass
Spectrometry
mass indicates
enzyme activity
Figure 1. Outline of the PECAN technology. The Nanostructure-Initiator Mass Spectrometry (NIMS) surface allows for high-throughput enzyme activity
determination in complex samples, circumventing chromatographic separations by means of in situ fluorous affinity purification of perfluorinated analytes. In
PECAN, a perfluorinated tag is covalently attached to the enzyme substrate and product(s) using Cu(I)-catalyzed “click” chemistry, after completion of the
enzyme reaction. Following the cleanup step, retained analytes are analyzed directly by NIMS.
µM NADP+ and 0.1 unit/mL G6P dehydrogenase). After a 3-hour
enzymatic reaction, the lysates were tagged with a perfluorinated alkyne
As a model high-throughput enzyme screening campaign, we aimed
to identify mutants of P450BM3 with the ability to oxidize valencene (3a)
to the fragrance and insect repellent nootkatone (5a)[33], a commonly-
studied biocatalytic process[34]. In such a process, a P450 hydroxylates
3a, producing either epimer of 4a. The P450 then again oxidizes the
same position on 4a to yield 5a (Fig. 3a). While wild-type P450BM3 is
unable to effect this transformation, mutants displaying this ability have
been discovered in studies evaluating small, focused mutant
libraries[35,36]. Although several P450BM3 variants were found to act on
3a, most are either unable to oxidize it beyond 4a, or oxidize 3a at more
than one position, yielding non-specifically over-oxidized products
(Figs. 3a, S4). Although PECAN cannot distinguish between isomers, it
can estimate the extent of probe oxidation from its mass distribution, and
thereby help reveal the library’s most promising members. While peak
heights of different ions in a mass spectrum cannot be directly quantified
in the absence of isotopically-labeled internal standards, we expect that
the analytes’ desorption and ionization in the PECAN assay is largely
determined by the tag, which is shared by all analytes. Regardless, the
MS signal of an ion proportional to all other tagged ions is expected to
rise monotonically with its concentration, allowing us to identify the
most productive wells in a screen based on ion counts.
through
a copper(I)-catalyzed “click” reaction, and acoustically
transferred onto a NIMS surface. The surface was washed with water
and rastered on a MALDI-TOF mass spectrometer. The resulting MS
image showed excellent signal-to-noise (Fig. 2c), and P450BM3 reactions
could easily be distinguished from negative controls both qualitatively
and quantitatively (Fig. 2d). The Z-factor – a commonly-used statistical
measure of the quality a high-throughput assay[32] – was calculated to be
0.93, indicating an excellent assay. Similar results were obtained for
biological replicates assayed in 96-deepwell microtiter plate format (Fig.
S1), suggesting that PECAN can be applied to high-throughput enzyme
screening experiments.
We also investigated whether PECAN could be used to monitor
intracellular enzymatic reactions. Probing enzymatic activity in vivo
rather than in lysate increases the relevance of screens to downstream
whole-cell bioconversion applications, could improve experimental
throughput by avoiding the lysis procedure, and decreases assay costs
by avoiding the need for exogenous cofactors. Using PECAN, we could
successfully monitor the oxidation of 1b fed to whole E. coli cells
expressing P450BM3 (Fig. S2).
Eukaryotic P450s are typically membrane-bound and studied in
microsomes (membrane fractions derived from the endoplasmic
reticulum), prompting us to test the applicability of PECAN to the
measurement of P450 activity in microsomes. We subjected the
contraceptive medication 19-norethindrone to recombinant human
microsomal P450 CYP3A4 and were able to detect its oxidation
products (Fig. S3). Unlike 1b, which contains an azide, 19-
norethindrone harbors an alkyne, requiring a reversal of the polarity of
the tagging “click” reaction. This did not noticeably affect the efficiency
of the tagging reaction or MS signal. CYP3A4 activity could be
abolished in the presence of the known inhibitor clotrimazole (Fig. S3),
suggesting that that PECAN may be suitable for drug-drug interaction
screening.
We performed combinatorial site-saturation mutagenesis (NNK
codons) on two P450BM3 amino acid residues commonly mutagenized
due to their proximity to the active site heme: F87 and A328[30,37]. While
a subset of this library has been assayed for 3a oxidation before[36], no
study has further explored its amino acid space, likely due to limited
experimental throughput. A lack of library sequence bias was verified
by Sanger sequencing 10 randomly-picked colonies. Like others, we
observed color differences between the different wells of our P450BM3
library (Fig. S5), which has been attributed to the ability of some variants
to oxidize indole to form the dyes indigo and indirubin[38]. Throughout
our study, we found no clear correlation between colored wells and the
enzyme’s ability to oxidize 3a.
Employing 3b as our PECAN probe analog of 3a, we screened 1208
E. coli cell lysates generated from our P450BM3 library for the ability to
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oxidize 3b into products with the mass of 5b, in a 96-well format. Hits
could be visually identified from the resulting MS image (Fig. 3c). For
a quantitative analysis, we used the OpenMSI Arrayed Analysis
Toolkit[39] to identify wells displaying m/z = 869 (i.e., 5c) relative ion
intensities more than 10 standard deviations above eight GFP controls
included on the same 96-well plate. These hits were de-replicated by
Sanger sequencing to yield the variants listed in Table 1. The identified
amino acid substitutions consist mostly of conservative small and
hydrophobic residues. Several variants were recovered more than once.
All variants were found to have 3b-oxidation activity in a confirmatory
PECAN experiment (Table S1).
can identify enzymes with desired catalytic activities that could
previously be discovered only using low-throughput approaches.
However, despite our ability to screen
a significantly more
comprehensive mutant library using the PECAN technology, we still
identified F87A/A328I as the optimal nootkatone-producing P450BM3
variant, suggesting that the “enriched library” strategy employed by
Seifert et al. was indeed effective. Other amino acid substitutions
yielding variants with substantial nootkatone production are S, T, Y, N
and G. Glycine, in particular, appears in many highly-active variants,
suggesting that future “enriched” site-saturation enzyme libraries may
benefit from including this amino acid.
Because the PECAN screen could not distinguish between isomers,
and because 4a is merely a surrogate substrate, we used GC-MS to assess
the ability of P450BM3 variant hits to oxidize 3a in vitro (Table 1).
Purified enzymes were employed to control for differences in protein
expression levels. Nearly all screen hits were able to catalyze the
conversion of 3a to 5a to some extent, whereas wild-type P450BM3
cannot. P450MB3 F87A, F87G, F87I, F87P and F87G/A238G were found
to be hyper-active non-specific 3a oxidizers, which is unsurprising
considering the small amino acid residues lining these variants’ active
sites. Three variants did not produce 5a, of which only one variant
(F87V/A238P) was completely inactive on 3a.
Two of our hits, F87A/A283I and F87A/A283V have previously
been identified by Seifert et al, who constructed an “enriched” library of
P450BM3 mutants substituting positions F87 and A328 with A, I, L, V
and F combinatorially (25 variants total) and tested for the ability to
convert 3a to 5a using GC-MS. This verifies that the PECAN technology
While the PECAN screen was able to identify P450BM3 variants
capable of producing nootkatone, some hits were false positives, and the
relative intensities of oxidation products observed using the PECAN
screen (e.g., 4c vs 5c) did not always match those measured by GC-MS
(e.g., 4a vs 5a, Table S1). This is likely because probe 3b is not a perfect
surrogate for 3a. Compared to 3a, which has 15 heavy atoms, 3b has 18,
presenting a 20% increase in size. Larger enzyme substrates, when
similarly functionalized with a “clickable” functional group, would be
expected to yield probes more representative of their archetype. To
further improve hit identification, PECAN could be used in conjunction
with P450 fingerprinting, in which enzyme variants’ activities on
multiple probes are correlated to activities on the substrate of interest
measured for a subset of the library[40–42]. Alternatively, “label-free”
technologies enabling the tagging of unfunctionalized biomolecules[43,44]
promise to avoid this bias altogether.
a
c
b
803.2 (1c)
819.2 (2c)
803.2
770 780 790 800 810 820 830 840 850 770 780 790 800 810 820 830 840 850
m/z
m/z
100%
d
80%
60%
40%
20%
0%
GFP
P450-BM3
Z’ 0.93
In vitro reactions
Figure 2. Benchmarking of the PECAN technology. a) Wild-type cytochrome P450BM3 is known to catalyze the hydroxylation of 1a. b) An analog of 1a, 1b, was
used as the substrate probe for the PECAN experiment. Upon tagging 1b and its hydroxylation product 2b using Cu(I)-catalyzed “click” chemistry, 1c and 2c
are formed, respectively. c) MS image showing the spatial distribution of ions with m/z = 803 (green, 1c) and m/z = 819 (violet, 2c), generated using OpenMSI[39]
.
Each of the pixels’ two color intensities maps linearly to their respective ion’s percentile intensity in the MS image. Lysates of 3 E. coli cultures expressing GFP
(negative controls) and 3 cultures expressing P450BM3 (rows, alternating) were left to act on 1b for 3h. Each lysate underwent a tagging reaction, and 1 nL of
each reaction was printed onto a NIMS surface 7 times (columns). Inset mass spectra are single-pixel spectra representative of the spot. Distance between
spots (pitch) is 0.75 mm. d) Percent turnover (assuming 1c and 2c ionize similarly) was calculated from the spots’ 803 and 819 ion intensities (area under the
MS curve). Data points (representing individual spots) are grouped by enzymatic reaction (i.e., rows in c).
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a
b
throughput analytical technologies to verify the hits, as is routine in this
field. Like spectrometric assays, the PECAN technology requires the
synthesis of a specialized probe and enzymatic reactions are performed
in microtiter plates. Therefore, we expect PECAN to have roughly
equivalent throughput to – and act as a complementary method to –
spectrometric assays. We expect PECAN to be a valuable technology
for drug discovery or directed evolution campaigns that focus on
enzymes for which no high-throughput screen currently exists.
Table 1. Variants of P450BM3 discovered in the PECAN screen, how many
times each variant was discovered in the screen, and their ability to oxidize 3a
in vitro, as determined by GC-MS.
c
P450BM3
variant
Number
of hits
% 4a[a,b]
% 5a[a]
% Mis-
% Over-
oxidized[a,c] oxidized[a,d]
Wild-type
F87A/A238I[f]
F87G/A238V
F87P
0
2
6
2
1
1
2
1
1
2
1
3
1
1
1
1
6
3
2
2
0.5 ±0.1
7.5 ±0.6
9.1 ±0.7
7.6 ±0.2
7.2 ±0.7
7.5 ±0.2
7.9 ±1.3
7.2 ±0.4
5.1 ±0.5
4.3 ±0.8
9.6 ±0.6
2.3 ±0.2
3.8 ±0.1
4.1 ±0.9
3.5 ±0.6
1.4 ±0.6
1.1 ±0.3
ND
ND[e]
0.15 ±0.01
0.7 ±0.2
ND
13.7 ±0.2
1.9 ±0.2
6.7 ±0.1
59.9 ±2.8
48.8 ±3.2
43.1 ±5.9
0.6 ±0.1
ND
11.2 ±0.7 0.34 ±0.03
7.3 ±0.1
5.8 ±0.7
4.8 ±0.1
4.2 ±0.6
4.0 ±0.2
3.8 ±0.3
3.3 ±0.5
1.6 ±0.1
1.0 ±0.1
0.95 ±0.07
11.4 ±1.9
14.9 ±2.3
14.8 ±1.6
1.5 ±0.1
F87A
F87I
F87G/A238L
F87A/A238V[f]
F87G/A238S
F87G/A238N
F87V
Figure 3. a) The P450-catalyzed oxidation of 3a to produce 4a, 5a, and other
oxidized isoprenoids b) Analogously, the PECAN probe 3b, a surrogate for 3a,
may be expected undergo P450-catalyzed oxidations to form 4b, 5b and
similarly over-oxidized products. Upon tagging and MS analysis, ions
corresponding to 3c and its various oxidized forms are observed. Structures 4b,
4c, 5b, and 5c represent a variety of isomers that cannot be distinguished by
PECAN. “Tag” is identical to that shown in Fig. 2. c) MS image resulting from
the high-throughput PECAN screening campaign for ValHN3-oxidizing mutants
of P450BM3. The image shows ions with m/z = 855 (3c) in green, and m/z =
869 (5c) in violet. White pixels indicate both ions are present. Each of the pixels’
two color intensities maps linearly to their respective ion’s percentile intensity in
the MS images. Composite image of 3 MS imaging experiments shown.
0.41 ±0.05
2.0 ±0.1
10.8 ±1.1
2.2 ±0.2
3.9 ±0.6
1.2 ±0.2
5.6 ±0.5
F87T
0.48 ±0.05 0.26 ±0.08
F87G
8.9 ±0.4
0.38 ±0.07 0.03 ±0.05
0.21 ±0.03 0.4 ±0.2
32.0 ±0.3
ND
F87A/A238Y
F87I/A238L
F87G/A238P
F87G/A238T
F87G/A238G
F87A/A238P
F87V/A238P
While the potential for high-throughput NIMS-based analysis of
complex enzyme reaction mixtures has frequently been alluded to, thus
far all experiments describing the analysis of complex samples using
NIMS have been limited to small studies[8,9,12], and all successful high-
throughput NIMS experiments have been conducted with purified
protein in low ionic strength buffer, avoiding the need for in situ fluorous
affinity purification[43,45,46]. The screening campaign described here is
the first demonstration of a high-throughput NIMS-based analysis of
enzyme activity in a complex matrix.
0.07 ±0.06
ND
0.13 ±0.07 0.12 ±0.02
0.11 ±0.04 0.10 ±0.02
ND
ND
ND
ND
8.4 ±1.9
ND
64.4 ±13.0
ND
1.5 ±0.2
ND
In short, we have introduced the PECAN mass-spectrometric
enzyme activity assay, and demonstrated its suitability for high-
throughput screening of P450s in complex matrices such as cell lysates
and microsomes. While here we have demonstrated the applicability of
PECAN only to P450s, we believe that, after the appropriate method
development, this technology could be applicable to any enzyme (or set
of enzymatic reactions), that changes the mass of its substrate.
Additionally, it may be possible to monitor isomerization reactions
through the application of tandem MS, or enantioselective reactions
through the use of isotopically labelled substrates[47]. As expected from
any high-throughput assay, our PECAN screen yielded some false
positives, and we re-screened a small subset of the library using low-
ND
ND
[a] In vitro oxidation of 0.5 mM 3a by 1 µM purified P450BM3 variant (i.e., 0.2
mol% catalyst loading) and a cofactor generation system capable of producing
10 mM NADPH, in 1 h, as percent of total GC-MS peak area. Unreacted 3a
makes up the remainder. See Table S1 for a further breakdown [b] Sum of
diastereomers. [c] Sum of all other oxidation products with the same nominal
masses as 4a (220 Da) or 5a (218 Da) (See Fig. S4). [d] Sum of all oxidation
products with masses higher than 220 Da, which may include products not
derived from 4a or 5a. [e] Not Detected. [f] Previously reported[36] (note
corrigendum[48]).
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This work was funded by National Institutes of Health Awards
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Keywords: High-throughput screening • Mass spectrometry •
Enzyme assays • Biocatalysis • Cytochrome P450
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10.1002/anie.201901782
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Tristan de Rond*, Jian Gao, Amin
Zargar, Markus de Raad, Jack Cunha,
Trent R. Northen, and Jay D. Keasling*
Page No. – Page No.
m/z
m/z
A high-throughput mass
Enzymatic reaction
in cell lysate or other
complex matrix
Acoustic
printing onto
NIMS surface
In situ cleanup:
fluorous surface retains
only tagged probe
Nanostructure-
Initiator Mass
Spectrometry
Change in
mass indicates
enzyme activity
spectrometric enzyme activity assay
enabling the discovery of cytochrome
P450 biocatalysts
Easy as pecan pie: A high-throughput enzyme assay was developed, based on a
combination of click chemistry, fluorous affinity purification, and Nanostructure-Initiator
Mass Spectrometry. The utility of the technique, called PECAN, was demonstrated by
screening a library of cytochrome P450s for activity on valencene.
This article is protected by copyright. All rights reserved.