Two-fold Bioorthogonal Derivatization by Different Formylglycine-Generating Enzymes

Article abstract of DOI:10.1002/anie.201803183

Formylglycine-generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site-specific oxidation of a cysteine residue to the aldehyde-containing amino acid C^α-formylglycine (FGly). This non-canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine-generating enzyme (FGE) and the iron-sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.

Full text of DOI:10.1002/anie.201803183

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Two-fold Bioorthogonal Derivatization by Different Formylglycine
Generating Enzymes
Authors: Tobias Krüger, Stefanie Weiland, Georg Falck, Marcus
Gerlach, Mareile Boschanski, Sarfaraz Alam, Kristian M.
Müller, Thomas Dierks, and Norbert Sewald
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.201803183
Angew. Chem. 10.1002/ange.201803183
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http://dx.doi.org/10.1002/ange.201803183

Angewandte Chemie International Edition
10.1002/anie.201803183
COMMUNICATION
Two-fold Bioorthogonal Derivatization by Different
Formylglycine Generating Enzymes
[
1]
[2]
[3]#
[1]#
[2]
Tobias Krüger, Stefanie Weiland, Georg Falck, Marcus Gerlach, Mareile Boschanski,
[
2]
[3]
[2]
[1]
Sarfaraz Alam, Kristian M. Müller*, Thomas Dierks*, and Norbert Sewald*
[
23-28]
Abstract: Formylglycine generating enzymes are of increasing
interest in the field of bioconjugation chemistry. They catalyze the
site-specific oxidation of a cysteine residue to the aldehyde contain-
sively found in prokaryotes.
Unlike FGE, AtsB oxygen-
independently catalyzes FGly formation with a broader substrate
scope also accepting proline-free motifs. Some AtsB variants
even accept serine for FGly-generation (Figure 1a).
α
[29-32]
ing amino acid C -formylglycine (FGly). This non-canonical residue
can be generated within any desired target protein and subsequently
be used for bioorthogonal conjugation reactions. The prototypic
formylglycine generating enzyme (FGE) and the iron-sulfur protein
AtsB display slight variations in their recognition sequences. We
designed specific tags in peptides and proteins that were selectively
converted by the different enzymes. The combination of the different
tag motifs within a single peptide or recombinant protein enabled the
independent and consecutive introduction of two formylglycine resi-
The aldehyde tag methodology has been proven highly effi-
cient for the generation of site-specific ADCs with defined drug
[
1,33–36]
antibody ratios.
However, the introduction of different
payloads remains a challenge. In order to overcome this prob-
lem, we present a new aldehyde tag methodology making use of
two different recognition sequences selectively addressed by
different formylglycine generating enzymes. Along this line, we
also present the isolation of AtsB from Methanosarcina mazei
dues and the generation of heterobifunctionalized protein conjugates. (MM-AtsB), its characterization and application in bioconjugation.
The M. mazei sulfatase, endogenous substrate of MM-AtsB,
Bioconjugation chemistry is a subject of growing interest, espe-
contains the recognition sequence CTAGR with an alanine
instead of a proline. We assumed that this proline-free motif
might not be accepted by FGE and thus bioorthogonality be-
tween both enzymes could be achieved. Therefore, a combina-
tion of CTAGR for MM-AtsB with CTPSR for FGE and the con-
secutive application of both enzymes would allow for two-fold
modification of target proteins. We first intended to test this
predicted bioorthogonality by incorporating these two recognition
sequences into a single peptide chain and analyze the specificity
of MM-AtsB and FGE mediated FGly formation.
[
1–3]
cially for the preparation of antibody drug conjugates (ADCs).
Selective introduction of bioorthogonal functionalities into the
[
4–11]
protein represents the main challenge in this research area.
Enzymatic modification avoids complex manipulations of the
translational machinery and only requires a short amino acid
sequence in the protein of interest, which is recognized by the
enzyme to incorporate a bioorthogonal functionality or to con-
[
12,13]
nect the payload to the protein.
The formylglycine generating enzyme (FGE) is a valuable
[
14–16]
tool for bioconjugation.
It catalyzes the oxygen-dependent
The mm-atsb gene was coexpressed with the isc-operon
oxidation of a cysteine residue within the recognition sequence
from Azotobacter vinelandii which encodes proteins supporting
α
[17]
[23,24]
CXPXR in type-I-sulfatases to C -formylglycine (FGly).
This
iron-sulfur cluster biosynthesis.
Cells were cultivated in
recognition sequence can be introduced into recombinant pro-
minimal medium under micro-aerobic conditions followed by an
anaerobic purification protocol using Ni -sepharose chromatog-
[
6,18]
2+
teins and converted by FGE in vivo or in vitro.
Eukaryotic
and prokaryotic variants of FGE are known, and different catalyt-
raphy. Typically, 10 mg/L of soluble MM-AtsB were obtained,
which was purified to give concentrations up to 22 mg/mL (Fig-
ure 1b).
[
19–22]
ic mechanisms have been postulated.
In addition, the iron-
sulfur protein AtsB of the radical-SAM (S-adenosyl methionine)
protein superfamily is another FGly-generating system exclu-
Efficient in vitro conversion of cysteine within CTAGR by MM-
AtsB was achieved for substrate peptide P1 that contains addi-
tional auxiliary sequences flanking the core motif as present in
the M. mazei sulfatase (Figure 1c). Product quantification was
performed by MALDI-ToF-MS and integration of the mass sig-
nals generated by the cysteine (m/z 1580.7) and corresponding
FGly variant (m/z 1562.8, Figures S1B, C). Comparable ioniza-
tion efficiencies were verified by analyzing a 1:1 mixture of the
[
[
1]
2]
T. Krüger, Dr. M. Gerlach, Prof. Dr. Norbert Sewald,
Organische und Bioorganische Chemie, Fakultät für Chemie
Universität Bielefeld
Universitätsstraße 25, 33615 Bielefeld
E-mail: norbert.sewald@uni-bielefeld.de
Dr. S. Weiland, M. Boschanski, Dr. Sarfaraz Alam,
Prof. Dr. Thomas Dierks
2
short model peptide Ac-YLCTPSR-NH and its FGly analog by
MALDI-ToF-MS (Figure S1A). After 40 to 80 min 35-90% of the
cysteine residue within P1 had been converted, depending on
the substrate/enzyme ratio (Figure S1D). In order to determine a
Biochemie I, Fakultät für Chemie,
Universität Bielefeld
Universitätsstraße 25, 33615 Bielefeld
E-mail: thomas.dierks@uni-bielefeld.de
maximum reaction rate and a K
incubated with 2.5-25 μM P1. Using a Lineweaver-Burk plot a
rough Vmax of 10.4 nmol/min·mg and a K of 4.1 μM could be
M
value, 1 μM MM-AtsB was
[
3]
G. Falck, Prof. Dr. K. Müller
Zelluläre und Molekulare Biotechnologie, Technische Fakultät
Universität Bielefeld
Universitätsstraße 25, 33615 Bielefeld
E-mail: kristian.mueller@uni-bielefeld.de
M
extrapolated (Figures S1E, S1F). Thus, the kcat value of cysteine
modification in synthetic peptides by MM-AtsB (0.56 per min, at
3
0°C) is similar to that of AtsB from Klebsiella pneumoniae (1.14
#
Authors contributed equally
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Angewandte Chemie International Edition
10.1002/anie.201803183
COMMUNICATION
Figure 1: Purification and enzymatic characterization of AtsB from Methanosarcina mazei (MM-AtsB). a) The formylglycine generating systems FGE and AtsB
α
2
have slightly different recognition sequences for the generation of C -formylglycine (FGly) (XH : reducing agent). b) SDS-PAGE (12.5%) analysis (Coomassie
2
+
stain) of the purification of MM-AtsB (54 kDa) by Ni -sepharose (M: Marker; L: Lysate; FT: Flow through; W: Wash; E: Elution). c) Model peptides for the enzy-
matic characterization of MM-AtsB with recognition sequences specific for AtsB (red) and FGE (blue) with the core motifs given in bold.
-
1
min , at 37°C), but clearly higher than that of AtsB from Clostri-
(Figures 2c, S3). This can be assigned to a quasi-molecular ion
[M+H] of a y fragment which is generated from the (FGly)TAGR
-
1
[23, 37]
+
dium perfringens (0.048 min , at 25°C).
In order to investigate the biocatalytic selectivity of MM-AtsB,
peptide P2 was prepared comprising the 15-mer recognition
sequence of P1 together with the FGE motif CTPSR (Figure 1c).
We hypothesized that the specificity of MM-AtsB for the CTAGR
sequence might be high enough for a selective introduction of
one FGly residue into the peptide backbone (Figure 2a), while
FGE should significantly prefer the CTPSR motif.
P2 was converted by MM-AtsB and after 80 min incubation
time two MS peaks were observed and assigned to the sub-
strate peptide (m/z 2422.9) and the ‘mono-aldehyde’ derivative
derivative by cleavage C-terminally to FGly.
In parallel, P2 was converted by FGE from Mycobacterium
tuberculosis (MtFGE) to give the aldehyde species P2b at m/z
[
38,39]
2405.4 (Figure 2d).
MS/MS-analyses provided dominant y-
and b-fragment ions at m/z 587.7 and 1818.8, respectively,
originating from a (FGly)TPSR derivative (Figure 2e). Subse-
quently P2b was incubated with 0.5 equiv. of MM-AtsB to intro-
duce the second FGly. After 80 min of incubation approximately
70% was converted to the corresponding ‘di-aldehyde’ P2c (m/z
2387.4, Figures 2f, S2). Thus, a step-by-step introduction of two
FGly-residues into the peptide backbone was accomplished in a
bioorthogonal manner.
(m/z 2404.9, Figures 2b, S2). In order to prove the substrate
specificity of MM-AtsB, the FGly species were further investigat-
ed by MS/MS-experiments. We discovered that FGly-containing
peptides show a specific fragmentation at the FGly peptide bond
upon laser induced ionizations, thus forming distinct peptide
fragments. The predicted aldehyde derivative P2a gives rise to a
prominent fragment ion at m/z 1933.3 in MS/MS measurements
The 15-mer MM-AtsB motif of P1 and the dual tag of P2 were
attached to the C-terminus of the DARPin E01, which is directed
against the epidermal growth factor receptor (EGFR), yielding
the model proteins D1, D2 and D3, respectively (Figures 3a,
[
40–44]
S4).
1
00
100
80
60
40
20
0
a)
b)
c)
Ac CTAGR
CTPSR NH
2
AtsB
FGE
2404.9
(P2a)
8
6
4
2
0
0
0
0
0
P2
2422.9 (P2)
1
933.3
m/zcalc = 2405.1
m/zcalc) = 2405.2
+
([M+H]
[M+H] )
(
+
P2a Ac
TAGR
CTPSR NH
2
Ac CTAGR
TPSR NH
2
P2b
m/zcalc = 1934.0 ([M+H] )
+
FGE
AtsB
Ac
TAGR
TPSR NH
2
P2c
2380
2390
2400
2410
2420
2430
2440
600
800
1000 1200 1400 1600 1800 2000
m/z
m/z
d)
e)
f)
2
387.4
1
00
100
100
80
60
40
20
0
(P2c)
1
818.8
mm / z/ zca
c
+
+
( [( M[ M+ +H H] ]
ca
l
lc==2 23 38 87 7. 2. 1
))
8
6
4
2
0
0
0
0
0
80
60
40
20
0
2
405.4
(
P2b)
m/zcalc = 1818.8 (M )
+
m/zcalc = 2405.1
2423.4 (P2)
2405.4 (P2b)
+
(
m[ M/ z+ H] =) 2405.2
calc
+
(
[M+H] )
5
87.7
m/zcalc = 587.3 ([M+H]
+
)
2380
2390
2400
2410
m/z
2420
2430
2440
600
800
1000 1200 1400 1600 1800 2000
2380
2390
2400
2410
2420
2430
2440
m/z
m/z
Figure 2: a) Two FGly residues can be successively introduced into a model peptide exploiting the enzymatic preferences of different formylglycine generating
systems. b) Enzymatic modification of P2 by MM-AtsB. The FGly species P2a can be observed at m/z 2404.9. c) MS/MS (LIFT) analysis of P2a yielding a promi-
nent fragment ion at m/z 1933.3. d) Enzymatic modification of P2 by MtFGE yielding the aldehyde P2b at m/z 2405.4. e) MS/MS (LIFT) analysis of P2b gives rise
to a fragment ion at m/z 1818.8. f) The FGly-containing peptide that was previously prepared with MtFGE (see SI) was modified by MM-AtsB affording the corre-
sponding di-aldehyde species P2c (m/z 2387.4).
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Figure 3: FGly dual tag system in DARPin E01 constructs. a) Three different DARPin constructs D1, D2 and D3 contain the MM-AtsB and FGE recognition
sequences. b) Bioconjugation experiments: D1 and D2 were enzymatically modified first by MM-AtsB and fluorescently labeled by HIPS ligation with the carbox-
yfluorescein derivative 1 (HIPS-CF). Fluorescently labeled D2 was then further modified by MtFGE followed by PEGylation with 2 (HIPS-PEG2k). c) SDS-PAGE
analysis of fluorescently modified D1 and D2 (Coomassie stain and in-gel fluorescence detection). Residual labeling in the negative controls is due to endoge-
[45]
nous E. coli FGly generating system. d) SDS-PAGE analysis of fluorescently modified and pegylated D2 (Coomassie stain and in-gel fluorescence detection).
The constructs were analyzed by CD spectroscopy using the
tag-less DARPin E01 as a reference in order to address poten-
tial changes in stability and folding. The CD spectra suggest
negligible impact of the recognition sequences on the DARPin
secondary structure (Figure S5).
2 days at 22 °C and pH 6.0, and the success of the conjugation
reaction was confirmed by SDS-PAGE and in-gel fluorescence
imaging (Figures 3b, c). Afterwards, the conjugates were purified
by anion exchange chromatography to remove remaining HIPS
reagent and MM-AtsB. The fluorescently labeled DARPin D2
(D2a) was subsequently incubated with MtFGE followed by a
second HIPS ligation with HIPS-PEG2k (2, Figure 3b). The reac-
tion products were separated by SDS-PAGE to evaluate the
success of the two-fold conjugation. D2a which had been incu-
bated with MtFGE prior to the addition of 2 showed a significant
mass shift from 20 to 25 kDa (Figure 3d), indicating that a sec-
ond payload was attached in a bioorthogonal way. The overpro-
portional increase of the molecular mass can be explained with
[
46]
The enzymatic conversions of D1, D2, and D3 by MM-AtsB
were performed at a 1:1 to 2:1 DARPin/enzyme ratio for 2 h at
30 °C. In order to confirm FGly formation, the reaction mixtures
were treated with trypsin either directly or after separation by
SDS-PAGE and were analyzed by MALDI-ToF-MS. The data
confirm that MM-AtsB accepts the CTAGR motif within D1 and,
furthermore, nearly exclusively modifies CTAGR, but not CTPSR
in D2 and D3 (Figures 3b, S6-9). Some minor conversion of the
CTPSR motif was observed only in the presence of higher con-
centrations of MM-AtsB.
[
47]
the larger hydrodynamic radius of the attached PEG2k moiety.
In order to assess whether the CTAGR-tag or the enzymatic
conversion by MM-AtsB would impair the ability of the DARPin
to bind EGFR, cell binding assays with fluorescently labelled D1
(D1a) by confocal microscopy and flow cytometry were carried
out. The microscopy experiments showed distinct accumulation
of fluorescent dye on the EGFR-rich surface of A431 cells (Fig-
ure S10A). Binding of D1a was not observed for MCF7 cells due
to their much lower expression level of the receptor. These
results were validated by flow cytometry analyses in which A431
cells showed a higher fluorescence signal shift after incubation
with D1a compared to MCF7 cells (Figure S10B).
The enzymatic conversions of D2 with FGE were performed
at a 10:1 of DARPin/enzyme ratio for 1.5 h at 25 °C. Besides M.
tuberculosis FGE, the homolog from Streptomyces coelicolor
was used which is known to display a more stringent substrate
[
38]
specificity for proline-containing motifs. However, under the
aforementioned reaction conditions, both enzymes almost ex-
clusively converted cysteine within CTPSR (Figure S7).
Proof-of-concept bioconjugation reactions were initiated with
the enzymatic modification of D1 and D2 by MM-AtsB followed
by the FGly-specific Hydrazino-iso-Pictet-Spengler ligation
[
35]
(
HIPS ligation, Figure 3b). The FGly-containing DARPins were
In summary, we present the first dual aldehyde tag motif
which incorporates two different recognition sequences that can
incubated with the carboxyfluorescein derivative 1 (HIPS-CF) for
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Angewandte Chemie International Edition
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COMMUNICATION
be selectively modified by different formylglycine generating
enzymes. AtsB from Methanosarcina mazei (MM-AtsB) nearly
exclusively converts the CTAGR motif within a dual tag that also
contains a classical CTPSR site. In contrast, FGE almost exclu-
sively converts cysteine within CTPSR. We incorporated the
newly designed tag sequences into the DARPin E01 for proof-of-
concept bioconjugation experiments. The results indicate that
the consecutive enzymatic conversion by different formylglycine
generating enzymes allows for a successive introduction of two
distinct payloads into a target protein. Moreover, the model
protein retains its function despite the addition of the tag and the
enzymatic transformation.
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Keywords: bioconjugation • enzyme catalysis • formylglycine •
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Angewandte Chemie International Edition
10.1002/anie.201803183
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Tobias Krüger, Stefanie Weiland, Georg Falck,
Marcus Gerlach, Mareile Boschanski,
Sarfaraz Alam, Kristian Müller*, Thomas Dierks*,
and Norbert Sewald*
Page No. – Page No.
Two-fold Bioorthogonal Derivatization by
Different Formylglycine Generating Enzymes
Alam
The formylglycine (FGly)-based bioconjugation strategy was expanded by utilizing the iron-sulfur protein AtsB. Combined
with the prototypic formylglycine generating enzyme (FGE) the independent introduction of two aldehyde moieties into
peptides and proteins was accomplished. This improved enzymatic system represents a new strategy for dual protein label-
ing at two specific sites.
This article is protected by copyright. All rights reserved.