Biocatalytic Reversal of Advanced Glycation End Product Modification

Article abstract of DOI:10.1002/cbic.201900158

Advanced glycation end products (AGEs) are a heterogeneous group of molecules that emerge from the condensation of sugars and proteins through the Maillard reaction. Despite a significant number of studies showing strong associations between AGEs and the pathologies of aging-related illnesses, it has been a challenge to establish AGEs as causal agents primarily due to the lack of tools in reversing AGE modifications at the molecular level. Herein, we show that MnmC, an enzyme involved in a bacterial tRNA-modification pathway, is capable of reversing the AGEs carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML) back to their native lysine structure. Combining structural homology analysis, site-directed mutagenesis, and protein domain dissection studies, we generated a variant of MnmC with improved catalytic properties against CEL in its free amino acid form. We show that this enzyme variant is also active on a CEL-modified peptidomimetic and an AGE-containing peptide that has been established as an authentic ligand of the receptor for AGEs (RAGE). Our data demonstrate that MnmC variants are promising lead catalysts toward the development of AGE-reversal tools and a better understanding of AGE biology.

Full text of DOI:10.1002/cbic.201900158

Accepted Article
Title: Biocatalytic Reversal of Advanced Glycation End Product
Modification
Authors: Nam Y Kim, Tyler N Goddard, Seungjung Sohn, David A
Spiegel, and Jason Crawford
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10.1002/cbic.201900158
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Biocatalytic Reversal of Advanced Glycation End Product
Modification
Nam Y. Kim,[a, b] Tyler N. Goddard,[a, b] Seungjung Sohn,[a] David A. Spiegel,[a, c] and Jason M.
Crawford*[a, b, d]
Abstract: Advanced glycation end products (AGEs) are
a
contributor of AGE-associated chronic inflammation and cellular
damage.[5] Elevated levels of AGEs are linked to the pathology of
many metabolic and degenerative diseases of aging, such as
diabetic complications, atherosclerosis, and Alzheimer’s
disease.[6-13] This association is manifested by age-dependent
increases in cross-linking, browning, fluorescence, and AGE
content in long-lived proteins such as collagens and lens
crystallins.[14-17] Structural characterization and synthesis of some
of the more prevalent AGEs (e.g., glucosepane) have allowed
more focused investigations into their individual chemical
properties and formation.[18] Indeed, chemical studies have shown
strong correlations between specific AGEs and the development
of age-related illnesses;[19,20] however, it has been difficult to
unequivocally demonstrate that any AGEs are direct causal
factors largely due to the lack of tools for investigating the reversal
of mature AGE modifications at the molecular level.
Previous correlative strategies to probe the biological roles of
AGEs have mainly focused on inhibiting their formation through
chemical interventions. For example, aminoguanidine can
intercept reactive α-dicarbonyls, including the AGE substrates
glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone
(3DG).[21] Additionally, molecules such as ALT-711 were
developed to “break” dicarbonyl protein crosslink intermediates
en route to mature AGEs, which showed favorable therapeutic
effects, including a reduction in collagen crosslinking and
myocardial stiffness in animal models.[22] More recently, enzymes
known as the Amadoriases and DJ-1 have been discovered to be
able to “repair” glycated proteins and nucleic acids,
respectively.[23,24] Although these enzymes are promising tools in
AGE research, they only act on the early intermediates in
glycation rather than mature AGEs. Currently, there are no known
strategies available to repair mature AGE modifications and
potentially ameliorate established AGE-associated pathologies.
Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine
(CML) are lysine-derived AGEs that have been extensively
investigated in renal compartments, collagen, and lens.[25]
Formation of CEL and CML involves the reaction between the ε-
amino group of lysine residues and MGO or GO, respectively.
Alternatively, CML is also known to form from the oxidative
cleavage of the Amadori product fructoselysine.[26] As two of the
major physiological ligands of RAGE,[27,28] CEL and CML are
associated with the progression of a host of diabetic complications
and other neurodegenerative diseases. Studies have shown
increased levels of CEL in serum samples of Alzheimer’s disease
patients, and reduction in permeability of the vitreous humor in the
eye.[29,30] As one of the most abundant AGEs in renal
compartments, CML was shown to be associated with a decline
in renal function and chronic kidney disease, although it is still
under debate whether or not CML is the causative agent[31-34] Here,
we describe the identification and characterization of an enzyme
family – MnmC – that is capable of cleaving CEL and to a lesser
heterogeneous group of molecules that emerge from the
condensation of sugars and proteins via the Maillard reaction. Despite
a significant number of studies showing strong associations between
AGEs and the pathologies of aging-related illnesses, it has been a
challenge to establish AGEs as causal agents primarily due to the lack
of tools in reversing AGE modifications at the molecular level. Here,
we show that MnmC, an enzyme involved in a bacterial tRNA-
modification pathway, is capable of reversing the AGEs carboxyethyl-
lysine (CEL) and carboxymethyl-lysine (CML) back to their native
lysine structure. Combining structural homology analysis, site-
directed mutagenesis, and protein domain dissection studies, we
generated a variant of MnmC with improved catalytic properties
against CEL in free amino acid form. We show that this enzyme
variant is also active on a CEL-modified peptidomimetic and an AGE-
containing peptide that has been established as an authentic ligand
of the receptor for AGEs (RAGE). Our data demonstrate that MnmC
variants are promising lead catalysts toward the development of AGE-
reversal tools and a better understanding of AGE biology.
Introduction
Advanced glycation end products (AGEs) are non-enzymatic
post-translational modifications of proteins derived from the
condensation of reducing sugars and nucleophilic amino acid
residues, such as lysine and arginine.[1,2] Although AGEs are
formed in the body as a part of normal metabolism, they can
accumulate to high concentrations and contribute to the
progressive decline of multiple organ systems.[3] This process is
accelerated in diabetics, owing to their hyperglycemic
conditions.[4] In addition to causing spontaneous damage by
altering protein structure and function, AGEs also interact with the
receptor for AGEs (RAGE), eliciting oxidative stress and
activating the transcription factor NF-B thought to be a major
[a]
N. Y. Kim, T. N. Goddard, S. Sohn, Prof. D. A. Spiegel, Prof. J. M.
Crawford
Department of Chemistry, Yale University
225 Prospect Street, New Haven, CT 06511 (USA)
N. Y. Kim, T. N. Goddard, Prof. J. M. Crawford
Chemical Biology Institute, Yale University
600 West Campus Drive, West Haven, CT 06516 (USA)
Prof. D. A. Spiegel
Department of Pharmacology, Yale School of Medicine
333 Cedar Street, New Haven, CT 06520 (USA)
Prof. J. M. Crawford
[b]
[c]
[d]
Department of Microbial Pathogenesis, Yale School of Medicine
333 Cedar Street, New Haven, CT 06536 (USA)
E-mail: jason.crawford@yale.edu
Supporting information for this article is given via a link at the end of
the document.
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extent CML modifications to restore the native lysine structure in
vitro. MnmC naturally functions in macromolecular RNA
modification as both an oxidase and a methyltransferase with Km
values of 600 ± 200 nM and 70 ± 40 nM for oxidation and
methyltransferase activities, respectively.[35] However, here we
show that the oxidase domain of the enzyme exhibits promiscuity
for the AGE substrates at higher concentrations. Through
homology analysis, site-directed mutagenesis, and protein
domain dissection studies, we generated variants of the enzyme
with an enhanced capacity to cleave CEL. We also demonstrate
that the enzyme not only cleaves CEL in free amino acid form, but
also cleaves CEL in both peptidomimetic and physiologically-
relevant peptide contexts. To the best of our knowledge, this study
provides the first biochemical characterization of a biocatalyst that
is capable of reversing a mature AGE in a peptide context. MnmC
variants could serve as directed evolution leads toward the future
development of new molecular AGE tools with improved kinetic
parameters for CEL and CML reversal.
Results
Figure 1. A) Growth of E. coli lysine auxotroph in M9 minimal medium
supplemented with lysine, CML, CEL, or water vehicle control. B) Identification
of genes required for CEL-cleavage through transposon mutagenesis.
Identification of a CEL-breaking biocatalyst
To assess whether E. coli was capable of cleaving the Nε-C bond
in CEL or CML to restore Lys, we fed free CML or CEL as a sole
Lys source to an E. coli Lys auxotroph (ΔlysA). E. coli ΔlysA was
cultivated in minimal M9 medium supplemented with 1 mg/mL Lys
(positive control), CML, CEL or water vehicle (negative control) at
30 oC overnight under aerobic conditions. Strong growth was
observed only for cultures supplemented with either Lys or CEL,
indicating that E. coli can utilize CEL as a sole Lys source (Figure
1A). To identify the biocatalyst(s) responsible for CEL cleavage,
we performed transposon mutagenesis in E. coli ΔlysA and
screened for mutants with impaired growth using CEL as a source
of Lys (Figure 1B). One mutant from our non-saturating
transposon mutant library (~3000 mutants were analyzed)
exhibited impaired growth. The transposon insertion site was in
mnmG (a.k.a., gidA), a gene located in the bacterial MnmEG
pathway responsible for wobble U34 tRNA modifications.[36,37]
However, isolated MnmG was not able to cleave CEL in vitro (data
not shown). Analysis of the broader pathway led us to further
study MnmC, an enzyme that catalyzes a tRNA modification
reaction analogous to CEL cleavage (Figure 2A, Figure 2B).[38]
Figure 2. Analogy between the transformation of cmnm5U34 to nm5U34 by C-
terminal domain of MnmC (C-MnmC) and the restoration of lysine from CEL.
A) Two consecutive reactions catalyzed by the bifunctional tRNA-modifying
enzyme MnmC in E. coli. C-MnmC catalyzes FAD-dependent removal of
carboxymethyl group in cmnm5U34 that generates a primary amine which is
methylated by SAM-dependent N-MnmC. B) Lysine restoration from CEL
cleavage. In both transformations, removal of carboxymethyl- or carboxyethyl-
functionality generates a primary amine.
Characterization of MnmC and validation of its activity on
CEL
MnmC is a bifunctional enzyme that is known to catalyze two
steps in the biosynthesis of the 5-methylaminomethyl-2-
thiouridine (mnm5(s2)U34) nucleoside in tRNA. The C-terminal
domain (C-MnmC) is an FAD-dependent oxidase that catalyzes
removal of the carboxymethyl functionality in cmnm5U34 to
generate a primary amine, and the N-terminal domain (N-MnmC)
is a SAM-dependent methyltransferase that catalyzes methylation
of this primary amine in nm5U34 to yield mnm5U34 (Figure 2A).
These domains are fused by an interdomain linker which, when
cleaved, yields the two domains in isolation that still retain
catalytic activity.[35,39] The C-MnmC domain-catalyzed removal of
a
carboxymethyl functionality to a primary amine in a
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0.9
0.6
0.3
40000
30000
20000
10000
0
CEL + MnmC
MnmC
CEL
Pyruvic Acid
10 mM CEL + MnmC
50 M Pyruvic Acid
1 mM CEL + MnmC
10 mM CEL Only
0.0
0
1
2
3
4
5
6
400
500
600
700
Retention Time (Min)
Wavelength (nm)
Figure 3. Validation of C-MnmC-mediated CEL cleavage using an α-keto acid derivatization assay. A) Mechanism of deamination by D-amino acid oxidases (DAAO).
B) Proposed mechanism of CEL cleavage and the formation of 2,4-DNPH-pyruvic acid hydrazone adduct. *MnmC exhibits hydrogen peroxide scavenging activity.
C) Detection of CEL cleavage-dependent hydrazone adduct formation spectrophotometrically. The adduct appears red-brown in color and its absorbance spectrum
shows λmax at 445 nm, and a shoulder at 515 nm. D) LC-MS validation of MnmC-dependent generation of syn- and anti- isomers (*) of the hydrazone adduct (m/z
267, negative ion mode).
macromolecular target is analogous to the removal of a
carboxyethyl (or carboxymethyl) functionality in the desired CEL
(or CML) cleavage reaction (Figure 2B). D-amino acid oxidases
(DAAOs) are the closest structural homologs of C-MnmC.
Because DAAOs are FAD-dependent enzymes known to catalyze
the oxidative deamination of various amines and neutral D-amino
acids (e.g., glycine, sarcosine, D-alanine and D-proline) to give
their corresponding α-keto acids, ammonia, and hydrogen
peroxide (Figure 3A), we first assessed MnmC’s ability to turnover
CEL using an established spectrophotometric hydrogen peroxide
assay using horseradish peroxidase (HRP) and chromogenic
substrates.[40] Like DAAOs, we expected that MnmC-mediated
cleavage of CEL could involve oxidation of the Nε-C bond to
generate an imine, which is then hydrolyzed to yield pyruvic acid,
L-Lys, and hydrogen peroxide (Figure 3B). However, we did not
detect hydrogen peroxide production, as this product was
consumed by MnmC (see Figure S1 for MnmC-mediated
quenching of hydrogen peroxide). Thus, to demonstrate that
MnmC exhibits activity toward CEL, we then adapted an α-keto-
acid derivatization assay which can be used to monitor pyruvic
acid formation as a result of CEL cleavage (Figure 3B).[41] In short,
2,4-dinitrophenylhydrazine (2,4-DNPH) readily reacts with pyruvic
acid to produce the 2,4-DNPH-pyruvic acid hydrazone adduct
which has
a distinctive red-brown color and absorbance
maximum at 445 nm that can be monitored to measure the activity
of MnmC on CEL (Figure 3C). At initial CEL concentrations of 1
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6
5
4
3
2
1
0
4
3
2
1
0
WT C-MnmC
WT MnmC
i
i
n
0
5
10
15
a
c
i
t
o
i
e
s
n
e
T
L
S
V
F
Y
L
F
2
1
R
A
K
K
4
0
5
C
m
Q
W
a
r
e
l
3
2
1
4
0
8
0
0
5
4
4
W
2
e
i
v
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1
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0
0
0
0
1
c
s
e
3
3
5
3
Y
/
L
n
6
5
5
5
5
CEL (mM)
3
o
h
c
b
o
b
.
Y
Y
Y
Q
R
C
Y
Y
M
-
C
Y
C
/
m
n
L
i
y
1
.
V
1
X
s
m
u
l
2
T
2
a
5
5
.
W
.
Y
Y
P
P
0.6
0.4
0.2
0.0
40
30
20
10
0
***
100 M Glyoxylic Acid
80 M Glyoxylic Acid
100 mM CML + C-MnmC
100 mM CML only
CML only
CML + C-MnmC
350
400
450
500
550
600
650
Wavelength (nm)
Figure 4. A) Relative activities of MnmC variants. Standalone WT C-MnmC shows about 5-fold improvement in CEL-cleaving activity. B) Steady-state kinetics of
WT MnmC and WT C-MnmC showing concentration dependencies with CEL. C) Detection of CML cleavage-dependent 2,4-DNPH-glyoxylic acid hydrazone adduct
formation spectrophotometrically. The adduct appears orange in color and its absorbance spectrum shows λmax at 455 nm. D) Quantitation of the amount of glyoxylic
acid generated from CML cleavage by C-MnmC.
mM and 10 mM, we detected formation of 2,4-DNPH-pyruvic acid
hydrazone adduct in an MnmC-dependent manner. Formation of
2,4-DNPH-pyruvic acid hydrazone products (syn- and anti-
isomers) were further confirmed by LC-MS using authentic
standards (Figure 3D).
In addition to CEL, we also tested whether MnmC is active on
CML, which bears the carboxymethyl functionality similar to its
native tRNA substrate. Despite the resemblance, we did not
observe MnmC activity on CML at a 10 mM concentration,
presumably due to differences in nonstandard substrate binding.
This result is consistent with the observation that the E. coli Lys
auxotroph does not grow in the presence of CML as a sole Lys
source. Consistent with our overall findings, we did observe weak
CML turnover using an MnmC variant at higher substrate
concentrations (100 mM, see below).
a 2-fold increase in relative activities, and combinatorial double
mutants did not show further improvements (Figure 4A). We then
reasoned that since the reactions catalyzed by C-MnmC and N-
MnmC are thought to be kinetically tuned,[35] dissection and
analysis of the desired C-terminal monodomain could exhibit
improved properties. Thus, we cleaved off the N-terminal domain
using a cut site within the interdomain linker (between Leu250 and
Pro251), allowing isolated C-MnmC to be expressed in a
catalytically competent form. Compared to the previously isolated
C-MnmC,[39] our C-MnmC variant contains an N-terminal His6-tag
with a shorter linker. This isolated C-MnmC monodomain
construct exhibited about a 5-fold enhancement in relative activity
compared to the wildtype didomain MnmC (Figure 4A),
suggesting that the N-terminal domain could participate in quality
control. In addition to E. coli MnmC, we also tested the activities
of didomain MnmC homologs from related Gammaproteobacteria,
Photorhabdus asymbiotica, Vibrio cholerae, Photorhabdus
luminescens, and Xenorhabdus bovienii, and their activities
indicate that MnmC variants could more broadly catalyze CEL
cleavage (Figure 4A).
Structure-based engineering of MnmC
To enhance the activity of MnmC on CEL, we evaluated the X-ray
crystal structure of E. coli MnmC (PDB: 3AWI). We first generated
a cmnm5U34 binding model of MnmC, and identified 10 residues
around the bound substrate that could potentially be mutated to
better accommodate the longer hydrophobic alkyl chain of CEL
(Figure S2). Relative activities of site-directed mutants at these
positions were then compared using the α-keto acid derivatization
assay (10 mM CEL, 5 μM MnmC, 4 hours at 37 oC). Three single
residue mutants (Y312W, Y521L, and Y504K) showed only about
Following the relative analyses of MnmC variants, we
established the steady-state kinetics of wildtype didomain MnmC
and monodomain C-MnmC for CEL using the α-keto acid
derivatization assay and a 2,4-DNPH-pyruvic acid standard curve
(Figure 4B, Figure S3). The Km values for MnmC and C-MnmC
were comparable at 5.2 ± 1.3 mM and 4.3 ± 0.6 mM, respectively,
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Table 1. Kinetic parameters for CEL cleavage by MnmC and C-MnmC[a]
Km
(mM)
kcat
(min-1)
kcat / Km
(M-1 min-1)
MnmC
5.2 ± 1.3
4.3 ± 0.6
0.04 ± 0.004
0.18 ± 0.009
8 ± 1
C-MnmC
42 ± 6
[a] Reaction mixtures contained CEL (1-13 mM), MnmC or C-MnmC (25 μM),
and FAD (5 μM) in 50 mM Tris buffer (pH 8.0). Kinetic parameters were
calculated from GraphPad software (version 7.0) and are reported as mean
± SD. Experimental time points were run in duplicate.
whereas kcat values were 0.04 ± 0.004 min-1 and 0.18 ± 0.009 min-
1
,
respectively. These data indicate that the ~5.3-fold
improvement of C-MnmC is largely a function of kcat. Table 1
summarizes the kinetic parameters for CEL cleavage by MnmC
and C-MnmC.
With the improved C-MnmC monodomain construct, we
evaluated CML as a substrate. CML turnover was only observed
at a higher substrate concentration (100 mM CML, 20 μM C-MnmC,
4 hours, 37 oC). C-MnmC-catalyzed CML cleavage leads to
glyoxylic acid and lysine (Figure S4). Spectrophotometric
detection of 2,4-DNPH-glyoxylic acid hydrazone adduct was
monitored at 455 nm (Figure 4C) and quantified using a 2,4-
DNPH-glyoxylic acid standard curve (Figure 4D, Figure S5).
Substrate scope of C-MnmC
Figure 5. Comparison of the crystal structures of MnmC from E. coli (PDB:
3PS9, orange) and D-amino acid oxidase from human (PDB: 2DU8, grey). A)
Superposition of MnmC and D-amino acid oxidase. B) Active site of the C-
terminal domain of MnmC (indicated by red arrow). C) Active site of D-amino
acid oxidase (indicated by red arrow). FAD is shown in green.
To have utility in probing the biological roles of CEL and CML
AGEs, reversal biocatalysts must function on CEL/CML-modified
peptide substrates. Encouraged by the fact that MnmC natively
uses a macromolecular substrate like mature AGEs, we further
evaluated the crystal structures of E. coli MnmC versus human
DAAO. Superposition of the two crystal structures shows a high
degree of structural similarity between the C-terminal domain of
MnmC and DAAO (Figure 5A). However, when the surface
representations of the active sites for the two enzymes are
compared, it becomes apparent that C-MnmC exhibits a wide and
exposed active site whereas DAAO has a closed active site
(Figure 5B, Figure 5C). This difference likely arises from the fact
that MnmC acts on a macromolecular tRNA substrate, whereas
DAAO acts on small molecule substrates (e.g., D-amino acids).
The open active site in C-MnmC suggests that the enzyme could
also utilize CEL in peptide contexts.
We then proceeded to test C-MnmC for its activity in a peptide
context by synthesizing a short linear peptide substrate DEF-
(CEL)-ADE (Pept-CEL). Pept-CEL is a mature CEL-modified
peptide (Figure 6A) that is known to bind to the positively charged
cavity of the V domain of RAGE largely through its interaction with
the carboxyethyl functionality.[27,28] To detect activity of C-MnmC
on Pept-CEL, an overnight enzymatic reaction containing 16 mM
peptide substrate and 40 μM C-MnmC was analyzed by LC-MS.
Pept-CEL was modestly converted to DEF-K-ADE in the
presence of C-MnmC (Figure 6C). We performed HRMS/MS
experiments to sequence the substrate and product peptides to
validate C-MnmC-mediated CEL to Lys residue cleavage (Figure
7A, Figure 7B). These studies identified C-MnmC as the first
catalyst capable of reversing a mature AGE modification and
suggest that C-MnmC orthologs could serve as directed evolution
leads for further development.
To investigate the substrate scope of C-MnmC, we first
synthesized
diketopiperazine-CEL
(DKP-CEL)
and
diketopiperazine-CML (DKP-CML) as peptidomimetics of mature
CEL and CML AGEs. Diketopiperazines are cyclic dipeptides that
mimic protein beta turns.[42] Consistent with weak turnover
observed for CML in free amino acid form, under our experimental
conditions C-MnmC was only able to turnover peptidomimetic
DKP-CEL (Figure 6A) at a 10 mM substrate concentration (Figure
6B), suggesting that C-MnmC may cleave mature CEL residues
in peptides. Activity for DKP-CEL was ~15% relative to free CEL.
Discussion
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utilize CEL as a sole lysine source. Through a combination of
transposon mutagenesis, screening, synthesis, and protein
biochemical studies, we identify and characterize MnmC as an
enzyme capable of CEL cleavage and Lys restoration. While our
non-saturating transposon studies initially led to the identification
of mnmG, we were unable to show MnmG-mediated CEL
cleavage. This prompted us to examine the broader bacterial
MnmEG pathway. Previous reports on the MnmEG pathway
established that it is tightly controlled,[39] suggesting the possibility
that mnmG disruption could affect other genes in the pathway.
This led us to focus on MnmC, which catalyzes an analogous
reaction to CEL/CML cleavage.
Previous homology searches on MnmC revealed that its C-
terminal domain is structurally related to glycine oxidases (GOXs),
sarcosine oxidases (SOXs), and DAAOs.[43,44] As FAD-dependent
oxidases, these enzymes catalyze a similar enzymatic reaction
where an N-C bond is oxidized to an imine which is subsequently
hydrolyzed. Intriguingly, C-MnmC is known to catalyze a reaction
in which imine generation followed by hydrolysis results in a
decarboxymethylation to release a primary amine. Our homology
modeling efforts showed that C-MnmC is structurally closest to
the DAAOs. High structural homology between C-MnmC and
DAAO could explain the fact that CEL, which is in effect an N-alkyl
alanine, is a better substrate than CML, which is in effect an N-
alkyl glycine. DAAOs are known to accept glycine as a substrate,
albeit with much lower affinity than functionalized D-amino acids
(Table S2).[45] This is consistent with our observation that C-
MnmC, as a homolog of DAAOs, shows much weaker activity
toward CML than CEL in vitro and in cell culture. Additionally, in a
recent study, CML was reported to be converted to N-
1.5
1.0
0.5
0.0
e
L
E
L
t
E
a
r
C
C
t
-
s
e
e
P
b
K
D
r
u
F
S
o
N
DEF-K-ADE
DEF-(CEL)-ADE + C-MnmC
DEF-(CEL)-ADE Only
carboxymethylcadaverine
(CM-CAD),
N-
carboxymethylaminopentanoic acid (CM-APA), and N-
carboxymethyl-Δ1-piperideinium ion by probiotic E. coli strains. [46]
These data further support our cell culture observations and
suggest that despite structural similarities between CEL and CML,
their catabolism pathways in E. coli appear to be divergent.
The capability of C-MnmC to remove CEL from free,
peptidomimetic, and mature peptide contexts can likely be
attributed to the enzyme’s open and large active site. Similarly,
Amadoriases I and II, which are well-known FAD-dependent
oxidases capable of oxidizing the early Amadori product (e.g.,
fructoselysine), are also known to be functional on peptide
substrates with similar Km values (4.2 mM and 5.6 mM for
glycated-Lys and glycated-Lys-Phe, respectively).[47] Upon
examination of the active sites of these Amadoriases, it becomes
apparent that their active sites are also quite large, which could
explain how these enzymes are able to function on peptide
substrates unlike DAAOs and GOXs with small, buried active sites
(Figure S6). One exception is the DAAO from the yeast
Rhodotorula gracilis (RgDAAO). With an unusually large active
site, the RgDAAO is known to accept cephalosporin C and
0
10
20
30
40
50
Time (min)
Figure 6. Activity of WT C-MnmC on a peptidomimetic substrate DKP-CEL and
a peptide substrate Pept-CEL. A) Structures of DKP-CEL and Pept-CEL. B)
Activity of C-MnmC on DKP-CEL relative to its activity on free CEL. C) LC-MS
detection of C-MnmC-mediated cleavage of Pept-CEL and the release of DEF-
K-ADE (*).
Despite numerous studies establishing AGEs as contributing
factors to the progression of degenerative diseases, it has been a
challenge to establish causal relationships between them.[20] One
major challenge is an inability to reverse AGE modifications and
evaluate potential disease amelioration. Biocatalysts that are
capable of reversing specific mature AGEs could lead to a new
arsenal of molecular tools for the study of AGEs, and if successful,
to recombinant-enzyme therapies. However, no such tools
currently exist. Here, we found that an E. coli lysine auxotroph can
convert it to glutaryl-7-aminocephalosporanic acid,
intermediate in the enzyme-catalyzed synthesis of cephalosporin
antibiotics.[48] CEL/CML cleavage and cephalosporin
a key
C
inactivation mediated by these types of enzymes represent
alternative activities that could be exploited for non-canonical
functions.
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fitted with an electrospray ionization (ESI) source coupled to an Agilent
1290 Infinity HPLC system. Spectrophotometric measurements were
performed
using
a
Thermo
Scientific
NanoDropTM
2000c
Spectrophotometer. Protein modelling and active site comparisons were
done using MOE and Chimera 1.12.[49,50]
Materials: Nε-(carboxyethyl)lysine (CEL) was purchased from Focus
Synthesis, LLC. Nε-(carboxymethyl)lysine (CML) was purchased from
Chem-Impex International. EZ-Tn5TM <KAN-2>TNP TransposomeTM Kit
was purchased from Epicentre. Ultra-micro (8.5 mm window height)
cuvettes were purchased from BrandTech Scientific. Peptide Fmoc-
D(otbu)E(otbu)FK(ivDDE)AD(otbu)E(otbu)-wang resin was synthesized
by Biomatik.
Strains: E. coli lysine auxotroph (ΔlysA) harboring kanamycin resistance
gene (JW2806-1, Keio Collection) was obtained from Coli Genetic Stock
Center at Yale University. In order to utilize transposomes in EZ-Tn5TM
<KAN-2>TNP TransposomeTM Kit which also contain the kanamycin
resistance gene, we generated an E. coli lysine auxotroph strain sensitive
to kanamycin. To this end, we excised the kanamycin resistance gene in
JW2806-1 through FLP recombination using the plasmid pCP20 encoding
the FLP recombinase, which yielded
a kanamycin-sensitive lysine
auxotroph (JW2806-1flpout) that was used as the host strain in the
transposon mutagenesis studies.
Figure 7. Representative tandem mass spectra for pre- and post-cleavage of
CEL functionality by C-MnmC. A) HRMS/MS spectrum for pre-cleavage
substrate DEF-(CEL)-ADE showing fragments that contain CEL. B) HRMS/MS
spectrum for post-cleavage product DEF-K-ADE showing fragments that
contain lysine instead of CEL. Addition of carboxyethyl functionality on a lysine
side chain corresponds to an increase of 72 units in mass, and this difference
is observed between fragments detected in pre- and post-cleavage tandem MS
spectra.
Sole lysine source growth studies, transposon mutagenesis and
screening: To evaluate whether E. coli can grow in presence of CEL or
CML as sole lysine source, E. coli strain JW2806-1flpout was cultivated in
0.5 mL M9 minimal medium supplemented with 1 mg/mL lysine, 0.1% CEL,
or 0.1% CML at 30 oC overnight under aerobic conditions. The same E.
coli strain was then used as a host to generate a transposon mutant library
using the EZ-Tn5TM <KAN-2>TNP TransposomeTM Kit following
manufacturer’s protocol. Briefly, electrocompetent JW2806-1flpout cells
were electroporated using
1 μL of the EZ-Tn5 <KAN-2> TNP
Transposome. 1 mL of SOC medium was then added to the cells and
incubated on a 37 oC shaker for 1 hour to facilitate cell outgrowth. Dilutions
of the aliquots of the recovered cells were then plated on M9 minimal
medium agar plates supplemented with 50 μg/mL kanamycin and 1 mg/mL
lysine. Resulting colonies were then picked into 96-well plates containing
300 μL of M9 minimal medium supplemented with 0.1% CEL and 50 μg/mL
kanamycin, and cultured at 30 oC overnight under aerobic conditions.
Genomic DNA from transposon mutants that showed impaired growth was
then extracted using the DNeasy Blood and Tissue Kit (Qiagen), which
was used as template for arbitrarily primed PCR.[51] The amplified DNA
segments near the transposon insertion sites were sequenced by Yale
Keck Sequencing facility and subjected to BLAST analysis to identify the
disrupted gene.
Conclusion
In summary, our study has shown that C-MnmC is a biocatalyst
that can cleave mature CEL modifications to restore native lysine
structures. To the best of our knowledge, this is the first
biochemical demonstration of an enzyme that can reverse a
mature AGE-functionalized peptide. While the kinetic parameters,
which are similar to known Amadoriases, could be substantially
improved, C-MnmC variants represent lead catalysts for further
directed evolution and development. As MnmC natively acts on
nucleic acids, glycated DNA (e.g., carboxyethyl/carboxymethyl-
deoxyguanosine) may also be suitable substrates to test in future
studies. Such improved AGE-reversal tools could in principle
enable a better understanding of the biology of AGEs at the
molecular level, elucidate their direct roles in the pathogenesis of
age-related diseases, and serve as leads for recombinant
enzyme therapies.
MnmC expression and purification: N-terminally His6-tagged MnmC
was cloned and expressed as previously reported[35] with slight
modifications. In brief, DNA encoding MnmC was amplified from E. coli
BW25113 genomic DNA with primers MnmC-fw and MnmC-rv (Table S1),
which contain an NdeI site at the 5’-end and a BglII site at the 3’-end. Upon
gel extraction (NucleoSpin Gel and PCR Clean-up, Macherey Nagel), the
amplified sequence was cloned into pET28a (Novagen) expression vector
between NdeI and BamHI sites. N-terminally His6-tagged MnmC was then
expressed in E. coli BL21 (DE3) (1L of TB media), grown to an OD600 of
0.5 at 37 oC, induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside,
and further cultivated under aerobic conditions (250 rpm) overnight at 20
oC. Cells were lysed by sonication, and protein was purified from cell lysate
using affinity chromatography (Ni-NTA agarose, Qiagen). Purified protein
was then concentrated using an Amicon Ultra-15 centrifugal filter unit with
50 kDa cutoff, and prepared into glycerol stocks for storage at -20 oC. To
clone N-terminally His6-tagged C-MnmC (Pro251 to stop codon), primers
Experimental Section
General experimental procedures: Low-resolution electrospray
ionization-mass spectrometry (ESI-MS) data were collected on an Agilent
6120 Quadrupole LC/MS system equipped with a Phenomenex Kinetex
C18 (100 Å) 5-μm (4.6 x 250 mm) column. High-resolution (HR) ESI-MS
data were obtained using an Agilent iFunnel 6550 QTOF MS instrument
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C-MnmC-fw and C-MnmC-rv (Table S1) were used to amplify DNA from E.
coli BW25113 genomic DNA, and the protein was isolated using identical
purification steps as in MnmC before concentrating with Amicon Ultra-15
centrifugal filter unit with 30 kDa cutoff.
We thank the Yale Claude D. Pepper Older Americans
Independence Center (Pilot Grant to J.M.C. and D.A.S.), the
SENS Research Foundation (Grant to D.A.S.), the American
Diabetes Association Pathway to Stop Diabetes (Grant 1-17-
VSN-04 to D.A.S.), and Yale University for financial support.
Site-directed mutagenesis: MnmC mutants were generated via the
QuickChange mutagenesis method with the corresponding pairs of
primers shown in Table S1 in the Supporting Information. pET28a
construct containing MnmC gene was used as a template. All mutations
were validated by sequencing through Yale Keck Sequencing facility.
Keywords: Enzyme catalysis • oxidoreductases • protein
engineering • protein modifications
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
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Nam Y. Kim, Tyler N. Goddard,
Seungjung Sohn, David A. Spiegel,
Jason M. Crawford*
Page No. – Page No.
Biocatalytic Reversal of Advanced
Glycation End Product Modification
Advanced glycation end products (AGEs) are non-enzymatic post-translational
modifications of proteins that can accumulate to high concentrations and contribute
to the decline of body tissues. A lead biocatalyst has been discovered to reverse
the AGE modifications in vitro which could be further developed to enable a better
understanding of AGEs at the molecular level.
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