Transketolase A from E. coli Significantly Suppresses Protein Glycation by Glycolaldehyde and Glyoxal in Vitro

Article abstract of DOI:10.1021/acs.jafc.7b03183

Short-chained carbonyl species such as glycolaldehyde and its oxidized pendant glyoxal are highly reactive Maillard agents, leading to the formation of protein modifications. These advanced glycation endproducts have gained considerable interest as they have been linked to various pathologies in vivo. The ability of transketolase to use glycolaldehyde as a substrate suggested the possibility to modulate carbonyl-driven Maillard reactions. Model incubations with recombinant transketolase A from Escherichia coli in the presence of bovine serum albumin and glycolaldehyde indeed led to a decrease in glycolaldehyde concentrations paralleled by the enzymatic conversion to erythrulose. As a result, reversibly protein-bound glycolaldehyde and the major final endproduct N⁶-carboxymethyl lysine were significantly reduced by approximately 50%, respectively. Glycolaldehyde is easily oxidized to glyoxal in the presence of amines and oxygen. In the presence of transketolase, the lower amounts of glycolaldehyde therefore also strongly suppressed the formation of glyoxal specific arginine modifications, measured as 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline after acid hydrolysis.

Full text of DOI:10.1021/acs.jafc.7b03183

Article
pubs.acs.org/JAFC
Transketolase A from E. coli Significantly Suppresses Protein
Glycation by Glycolaldehyde and Glyoxal in Vitro
±
≠
,±
Alexander Klaus, Thorsten Pfirrmann, and Marcus A. Glomb*
±
Institute of Chemistry, Food Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 2, 06120 Halle/Saale,
Germany
≠
Institute of Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystr. 1, 06114 Halle/Saale, Germany
ABSTRACT: Short-chained carbonyl species such as glycolaldehyde and its oxidized pendant glyoxal are highly reactive
Maillard agents, leading to the formation of protein modifications. These advanced glycation endproducts have gained
considerable interest as they have been linked to various pathologies in vivo. The ability of transketolase to use glycolaldehyde as
a substrate suggested the possibility to modulate carbonyl-driven Maillard reactions. Model incubations with recombinant
transketolase A from Escherichia coli in the presence of bovine serum albumin and glycolaldehyde indeed led to a decrease in
glycolaldehyde concentrations paralleled by the enzymatic conversion to erythrulose. As a result, reversibly protein-bound
6
glycolaldehyde and the major final endproduct N -carboxymethyl lysine were significantly reduced by approximately 50%,
respectively. Glycolaldehyde is easily oxidized to glyoxal in the presence of amines and oxygen. In the presence of transketolase,
the lower amounts of glycolaldehyde therefore also strongly suppressed the formation of glyoxal specific arginine modifications,
measured as 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline after acid hydrolysis.
KEYWORDS: transketolase, glycolaldehyde, glyoxal, Maillard reaction, advanced glycation endproducts
6
INTRODUCTION
potent precursors of N -carboxymethyl lysine, the quantitively
■
5
predominant AGE in vivo. Surprisingly, literature on formation
Transketolase (TK, EC 2.2.1.1) is an ubiquitous enzyme
occurring in all organisms and of major importance in
carbohydrate metabolism, as it interconnects the pentose
phosphate pathway to glycolysis. The TK molecule is
of glycolaldehyde in vivo is scarce. Several studies proposed the
formation of glycolaldehyde during TK one-substrate-catalyzed
reactions. However, the release of free glycolaldehyde from the
enzyme was not detected. This might be explained by the
immediate condensation with another glycolaldehyde residue,
formed in the course of cleavage leading to erythrulose.
addition to the nonenzymatic Maillard induced generation of
glycolaldehyde, the myeloperoxidase system is the only known
enzymatic source for glycolaldehyde in vivo. Interestingly,
Thornalley et al. described the suppression of methylglyoxal,
due to a decrease in the triose phosphate pool via activation of
the TK catalyzed metabolization. As glycolaldehyde, methyl-
glyoxal represents another potent glycating agent in vivo and is
readily formed by the enzymatic and nonenzymatic degradation
of triose phosphates.
composed of two equal subunits, requiring thiamine1
diphosphate (ThDP) and bivalent cations for catalytic activity.
3
,7,8
In
The ThDP-mediated catalysis can be divided into two steps:
the cleavage of a C−C-bond of a donor ketose and subsequent
transfer of a covalently bound glycolaldehyde unit onto an
acceptor aldose. Xylulose-5-phosphate, fructose-6-phosphate,
and sedoheptulose-7-phosphate usually serve as donor
substrates and ribose-5-phosphate, glyceraldehyde-3-phosphate,
and erythrose-4-phosphate as acceptor substrates. TKs from
nonmammalian cells are also able to utilize unphosphorylated
sugars (e.g., β-hydroxypyruvate, dihydroxyacetone, and glyco-
laldehyde). In the case of β-hydroxypyruvate, the reaction
9
10
The aim of the present study was to elucidate the role of free
glycolaldehyde during two-substrate-catalyzed TK reactions
and the impact on the formation of AGEs. Special emphasis
was placed on the analysis of all mechanistic key intermediates
leading to stable protein bound endproducts during model
incubations with β-hydroxypyruvate and ribose-5-phosphate. As
a result, transketolase A (TKA) from E. coli lowered
concentrations of Schiff base adducts and thereby suppressed
becomes irreversible due to the release of CO . Apart from the
2
two-substrate reaction, TK is able to perform one-substrate
reactions, cleaving the donor substrate in the absence of an
acceptor aldose. However, the rate is substantially lower than
1
−3
during two-substrate-catalyzed reactions.
Short-chained aldehydes, like glycolaldehyde, play a major
role in the reactions leading to post-translational protein
modifications in vivo. Nonenzymatic carbonyl-amine reactions
are termed Maillard reactions, and the final reaction products
are also referred to as advanced glycation endproducts (AGEs)
6
synthesis of downstream modifications as N -carboxymethyl
lysine (CML) and 5-(2-imino-5-oxo-1-imidazolidinyl) norva-
line (imidazolinone).
4
−6
and accumulate over time.
Glomb et al. demonstrated that
glycolaldehyde is initially reversibly bound to proteins via Schiff
base adducts, but can be stabilized and analyzed by reduction as
Received: July 11, 2017
Revised: August 25, 2017
Accepted: August 29, 2017
6
5
N -(2-hydroxyethyl) lysine (HEL). Furthermore, their data led
to the conclusion that glycolaldehyde, next to glyoxal and the
Amadori product of lysine and glucose, is one of the most
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.7b03183
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Journal of Agricultural and Food Chemistry
Article
Inactivation of Transketolase. TKA was inactivated at 80 °C for
.5, 1, and 2 h. No activity was observed by above assay after 0.5 h.
Model Incubations. Incubations were conducted in HEPES buffer
at pH 7.4 and 25 °C. Ribose-5-phosphate and β-hydroxypyruvate were
incubated with TKA (0.06 mg/mL) and BSA (1 mg/mL) with and
without addition of glycolaldehyde (1 mM). Blank values were
obtained with inactivated TKA. The reaction mixture had the
following composition: 25 mM HEPES, 10 mM ribose-5-phosphate,
MATERIALS AND METHODS
■
0
Chemicals. All chemicals of the highest quality available were
provided by Sigma-Aldrich (Munich/Steinheim, Germany), Fluka
Taufkirchen, Germany), Merck (Darmstadt, Germany), Roth
Karlsruhe, Germany), and Sigma (Taufkirchen, Germany), unless
otherwise indicated.
(
(
HEL, CML, and imidazolinone were synthesized according to
5
,11
literature.
-(Naphthalene-1-ylamino)-ethanol (GA-N). GA-N was syn-
1
0 mM β-hydroxypyruvate, 3 mM MgCl , and 2 mM thiamine
2
2
diphosphate. Prior to incubations, TKA was rebuffered to 20 mM
HEPES. The reaction was initiated by the addition of TKA. Solutions
were degassed with helium and deaerated with argon before
incubation.
thesized according to ref 18. In the first step, 1.65 mmol of
glycolaldehyde was dissolved in 5 mL of water. An equimolar mixture
of 1-naphthylamine in 5 mL of 15% acetic acid and dimethyl sulfoxide
(
50:50, v/v) and a solution of 3 mmol sodium cyanoborohydride in 5
Analytical GC-FID. Analysis of trimethylsilyl derivatives of ribose-
mL of water were added and stirred at 40 °C overnight. The solution
was extracted with hexane (50 mL), and the organic phase was
concentrated under vacuum. Purification was carried out by column
chromatography (silica gel 60, hexane-acetone 7:3). The combined
5-phosphate, sedoheptulose-7-phosphate, and erythrulose was carried
1
7
out adopting the method of Smuda et al. To 100 μL aliquots of the
samples, 100 μL of D-sorbit (1 mM) was added and centrifuged with
MWCO 3000 at 5000 rpm, 4 °C. Samples were dried in vacuo,
residues were dissolved in anhydrous pyridine (100 μL), and N,O-
bis(trimethylsilyl)-acetamide with 5% trimethylchlorosilane (100 μL)
was added. Samples were sonified for 30 min and kept at room
temperature for 3 h prior to injection into the GC-FID system.
Samples were analyzed on a HP 6890N chromatograph (Agilent
Technologies, Palo Alto, CA) equipped with a flame ionization
detector. The column was a HP-5 (30 m × 0.32 mm, film thickness
0.25 μm; AgilentTechnologies, Palo Alto, CA); injector, 250 °C; split
ratio, 1:10; detector, 270 °C. Helium 4.6 was used as carrier gas in
constant flow mode (linear velocity 31 cm/s, flow 1.6 mL/min). The
oven temperature program was as follows: 100 °C, 5 °C/min to 200
fractions containing GA-N (TLC (silica gel): R 0.35, hexane/acetone
f
(
7:3), UV detection) were concentrated under vacuum to obtain the
compound as a brownish oil (0.86 mmol, 52%), which was identified
1
13
12
by comparison of its H and C NMR spectra reported in literature.
HR-MS: m/z 188.1070 (found); m/z 188.1070 (calculated for
+
C H NO [M + H] ).
1
2
14
Sedoheptulose-7-phosphate. Sedoheptulose-7-phosphate was
synthesized with TKA E. coli according to literature. Synthesis was
1
13
confirmed by comparison of its H and C NMR spectra to published
13
data. HR-MS: m/z 289.0333 (found); m/z 289.0330 (calculated for
−
C H O P [M − H] ). Purity was determined after derivatization by
7
14 10
°
C, 10 °C/min to 270 °C (held 10 min). Limit of detection and limit
of quantitation (LOD/LOQ) and retention times were as follows:
erythrulose (0.13/0.4 mol % ribose-5-phosphate) t = 9.72 min, sorbit
reductive amination as described below.
Protein Expression and Purification. The plasmid pGSJ427
carrying the gene for transketolase A from E. coli and His6-tag was
kindly made available by Dr. Ralph Golbik (University of Halle,
Germany). Expression and purification was carried out according to a
R
t = 19.35 min, ribose-5-phosphate (0.2/0.6 mol %) t = 22.10 and
R
R
2
2.27 min and sedoheptulose-7-phosphate (0.5/1.7 mol %) t = 25.74,
R
14,15
22.85, and 22.94 min. Quantitation was carried out by comparison of
peak areas obtained with those of standard solutions containing known
amounts of the pure authentic reference compounds. Signals of target
compounds were standardized using the signal of silylated sorbit.
Analytical HPLC-FLD. Determination of glycolaldehyde was
modified method as described in the literature.
Transformation of
the plasmid into E. coli Rosetta(DE3) (Novagen) strains was
performed by electroporation. The transformed cells were incubated
overnight on LB plates containing ampicillin 100 μg/mL and
chloramphenicol 35 μg/mL at 37 °C. A single colony was inoculated
into 300 mL of LB medium (100 μg/mL ampicillin, 35 μg/mL
chloramphenicol) and grown overnight. The culture was then
transferred to 4 L of fermentation medium (LB medium, 20 g
18,19
carried out following a modified method of Rakete and Glomb.
To 50 μL of the samples, a solution of 25 μL of 0.4 M 1-
naphthylamine (DMSO/15% acetic acid, 1:1, v/v) and 25 μL of 4 M
sodium cyanoborohydride (DMSO/water, 1:1, v/v) were added and
reincubated for 24 h. The derivatized samples were diluted with eluent
B to concentrations appropriate for detection. Eluents were
demineralized water (A) and a mixture of methanol and demineralized
water (7:3, v/v; B). 0.6 mL/L Heptafluorobutyric acid (HFBA) was
added to both eluents as ion pair reagent. Chromatographic
separations were performed on a stainless steel column (KNAUER,
Eurospher 100-5 C18, 250 × 4.6 mm, 5 μm, Berlin, Germany) using a
glucose, 2.7 g MgSO , 70 μM thiamine diphosphate (ThDP), 100 μg/
4
mL ampicillin, 35 μg/mL chloramphenicol) at 37 °C. At an OD600 of
5
, cells were harvested by centrifugation (10 000g, 15 min, 4 °C).
For protein purification, 15 g of cells was suspended in lysis buffer
(
50 mM NaH PO , pH 8.0, 300 mM NaCl, 1 mM MgCl , 10 mM
2
4
2
imidazole), and lysozyme (1 mg/mL) was added on ice. After 30 min,
cells were disrupted using a sonifier. Cell debris was pelleted by
ultracentrifugation at 10 000g for 20 min. The supernatant was applied
to a Ni2 NTA (Qiagen) column in a batch process and washed four
times with lysis buffer and equilibration buffer (50 mM NaH PO , pH
−1
flow rate of 1 mL min and a column temperature of 25 °C. Samples
+
were injected (25 μL) at 40% B; the gradient was then changed to
2
4
1
00% B within 15 min and held for 10 min. Then gradient was
8
.0, 300 mM NaCl, 1 mM MgCl , 20 mM imidazole), respectively.
2
changed to 40% B within 5 min, held 10 min. Glycolaldehyde
TKA was eluted stepwise with increasing concentrations of 125 mM
imidazole and 250 mM imidazole. Fractions containing TKA were
pooled, rebuffered to 20 mM glycyl-glycine with 1 mM dithiothreitol,
and concentrated to 1.65 mg/mL by ultrafiltration using micro-
concentrators (Vivaspin MWCO 30000, 4000 rpm, 4 °C) for storage.
Purity of isolated TKA was verified by SDS-Page, and protein
concentration was determined spectrophotometrically at 280 nm.
Transketolase Activity Assay. TKA activity was measured
spectrophotometrically according to a modified method of Hequet
naphthylamine derivative was detected at the retention time of t =
R
1
2.9 min with a LOD/LOQ of 0.006/0.02 mol % (ribose-5-
phosphate). Quantitation was performed using the standard addition
method with the pure authentic reference compound.
2
Analytical HPLC-MS . A Jasco PU-2080 Plus quaternary gradient
pump with degasser and a Jasco AS-2057 Plus autosampler (Jasco,
Gross-Umstadt, Germany) were used. The mass analyses were
performed using an Applied Biosystems API 4000 quadrupole
instrument (Applied Biosystems, Foster City, CA, U.S.A.) equipped
with an API source using an electrospray ionization (ESI) interface.
The LC system was connected directly to the probe of the mass
spectrometer. Nitrogen was used as sheath and auxiliary gas. To
measure the analytes, the scheduled multiple-reaction monitoring
16
et al. The reaction mixture (200 μL) contained 50 mM HEPES
buffer (pH = 7.4), 10 mM ribose-5-phosphate, 10 mM L-erythrulose, 3
+
mM MgCl , 2 mM thiamine diphosphate, 7.5 mM NAD , and
2
aldehyde dehydrogenase from S. cerevisiae (0.5 U) as auxiliary enzyme.
2
The reaction was initiated by addition of TKA. The reduction of
(sMRM) mode of HPLC-MS was used. Quantitation was based on
+
NAD was monitored in a 96-well plate at 340 nm on a Tecan Infinite
the standard addition method using known amounts of the pure
authentic reference compounds.
M200 plate reader at 25 °C. (Tecan, Groedic, AUT)
B
DOI: 10.1021/acs.jafc.7b03183
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Journal of Agricultural and Food Chemistry
Article
Figure 1. Transketolase-catalyzed metabolization of glycolaldehyde which reacts with BSA to form AGEs or alternatively serves as an acceptor aldose
to give erythrulose. Dashed arrows indicate alternative side-pathways.
Glyoxal. Sample aliquots were taken, and trichloroacetic acid was
added to a final concentration of 12.5%. o-Phenylenediamine (3 mM)
in 0.05 N HCl was added and reincubated for 5 h at 25 °C in the dark.
The derivatized samples were diluted with water to concentrations
appropriate for detection. The mobile phase used consisted of
methanol/water (7:3 (v/v)), with 0.6 mL/L HFBA. Chromatographic
parameters and optimized parameters for mass spectrometry were
gas. The sample solutions were introduced continuously via a 500 μL
Hamilton syringe pump with a flow rate of 5 μL min . The data were
evaluated by the Xcalibur software 2.7 SP1.
−1
Nuclear Magnetic Resonance Spectroscopy (NMR). NMR
spectra were recorded on a Varian VXR 400 spectrometer operating at
1
13
400 MHz for H and 100 MHz for C or on a Varian Unity Inova 500
1
13
instrument operating at 500 MHz for H and 125 MHz for C,
20
respectively. SiMe was used as a reference for calibrating the chemical
according to literature.
4
shift.
AGEs. Acidic hydrolysis after reduction with NaBH was carried out
4
5
Statistical Analysis. Analyses were performed in triplicate for each
model system and resulted in coefficients of variation less than 5% for
sugar and sugar phosphate concentrations and less than 10% for AGE
concentrations. All significance tests were performed by two-sample t-
Test or WELCH-Test with a probability value of 95%. Limit of
detection (LOD) and limit of quantitation (LOQ) were estimated
according to DIN 32645.
as described in literature. Chromatographic separations were
performed on a stainless steel column (VYDAC 218TP54, 250 ×
4
.6 mm, RP18, 5 μm, Hesperia, U.S.A.) using a flow rate of 1 mL
−
1
min and a column temperature of 25 °C. Aliquots of the samples
were diluted with water and injected (15 μL) at 2% B and run isocratic
for 15 min; the gradient was changed to 100% B within 15 min (held
for 10 min). Then the gradient was changed to 2% B within 5 min
(
held 15 min). Eluents were demineralized water (A) and a mixture of
methanol and demineralized water (7:3, v/v; B), with 1.2 mL/L
HFBA. For mass spectrometric detection, the sMRM mode was used,
optimized MS parameters were as follows: HEL (m/z 191.2/84.0 (DP
RESULTS AND DISCUSSION
■
Characterization of Transketolase A (TKA) and Model
Incubations. Approximately 1.7 mg of TKA per g of wet cells
was isolated with a specific activity of 0.2 U/mg. The
comparatively low activity of TKA is most likely due to the
use of a phosphate buffer during purification and an adopted
assay, as described in Materials and Methods. Complete
characterization and kinetic studies for TKA from E. coli were
4
2 V, CE 28.0 eV, CXP 6.0 V, quantifier), m/z 191.2/130.2 (DP 42 V,
CE 18.0 eV, CXP 23.0 V, qualifier), m/z 191.2/56.2 (DP 42 V, CE 56.0
eV, CXP 4.0 V, qualifier), imidazolinone (m/z 215.1/100.1 (DP 48 V,
CE 20.0 eV, CXP 8.0 V, quantifier), m/z 215.1/70.1 (DP 48 V, CE
3
8.0 eV, CXP 11.3 V, qualifier), m/z 215.1/116.2 (DP 48 V, CE 20.0
21
eV, CXP 9.0 V, qualifier) and CML. Retention times were as follows:
CML t = 6.2 min, HEL t = 10.0 min, and imidazolinone t = 13.8
22
R
R
R
performed previously by Sprenger et al.
min.
To investigate the influence of TKA on the formation of
AGEs, equimolar amounts of β-hydroxypyruvate and ribose-5-
phosphate with and without addition of 10 mol %
glycolaldehyde were incubated in the presence of bovine
serum albumin (BSA). As shown in Figure 1, the ThDP-
mediated initial cleavage of β-hydroxypyruvate results in the
Ninhydrin Assay. After complete workup, the content of amino
acid modification was standardized to L-leucine according to a
21
modified method of Smuda et al. A calibration of L-leucine,
concentrated between 0.5 and 10 nmol, was performed and referenced
to the diluted samples. Each sample was prepared in duplicate.
High-Resolution Mass Determination (HR-MS). Positive- and
negative-ion high-resolution ESI mass spectra were obtained from an
Orbitrap Elite mass spectrometer (Thermofisher Scientific, Bremen,
DEU) equipped with an HESI electrospray ion source (spray voltage 4
kV; capillary temperature 275 °C, source heater temperature 40 °C;
FTMS resolution > 30.000). Nitrogen was used as sheath and auxiliary
formation of CO and dihydroxyethyl-thiamine diphosphate
2
(
DHEThDP), the carbanion-enamine intermediate. The ThDP
bound glycolaldehyde unit is then transferred to ribose-5-
phosphate forming sedoheptulose-7-phosphate as the major
product. Addition of glycolaldehyde to the incubation mixture
C
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Journal of Agricultural and Food Chemistry
Article
Table 1. Mean Concentration (mol % ± Standard Deviation, Ribose-5-phosphate) of Sugar Phosphates, Sugars, and Glyoxal in
the Presence of BSA and Transketolase A (TKA) or Inactivated Transketolase A (ITKA) at 8 h of Incubation Time, with and
without Addition of 10 mol % Glycolaldehyde (GA)
TKA without GA
ITKA without GA
TKA with GA
ITKA with GA
a
c
sedoheptulose-7-phosphate
ribose-5-phosphate
glycolaldehyde
87.3 ± 1.6
11.5 ± 0.6
0.108 ± 0.006
0.18 ± 0.02
<LOD
82.0 ± 1.9
13.5 ± 0.5
<LOD
c
89.2 ± 0.7
0.536 ± 0.009
0.31 ± 0.02
88.5 ± 0.8
3.7 ± 0.1
1.17 ± 0.03
<LOQ
d
d
1.23 ± 0.03
c
d
glyoxal
0.34 ± 0.02
b
c
erythrulose
0.58 ± 0.05
<LOQ
5.3 ± 0.3
a
b
c
d
LOD: limit of detection. LOQ: limit of quantitation. Significant difference compared to incubations without GA by t-Test (α = 0.05). Significant
difference compared to incubations without GA by WELCH-Test (α = 0.05).
can either result (I) in the spontaneous formation of Schiff base
adducts with the amine moieties of BSA or (II) in the
formation of erythrulose by enzymatic condensation. β-
Hydroxypyruvate is of advantage as a donor substrate in this
model setup, as it renders the reaction irreversible. However, it
is also prone to spontaneous decomposition reactions leading
to glyoxylic acid, glycolaldehyde, and trace amounts of
irreversible enzymatic conversion of β-hydroxypyruvate and
ribose-5-phosphate to sedoheptulose-7-phosphate. The level of
sedoheptulose-7-phosphate quickly reached 82.0 mol % after 8
h and stayed nearly constant afterward. No formation of
sedoheptulose-7-phosphate was observed in the presence of
inactivated TKA. The concentrations of sugar phosphates,
erythrulose, glycolaldehyde, and glyoxal at 8 h of incubation are
summarized in Table 1.
erythrulose via nonenzymatic aldol condensation reac-
2
3−25
tions.
In order to exclude the possibility of a significant
Interestingly, given the high-glycating activity of glycolalde-
hyde, TKs have been shown to be relatively resistant to high
glycolaldehyde concentrations and therefore have been used in
impact on the formation of advanced glycation endproducts by
Maillard-catalyzed degradation of the initial products or
products formed during enzymatic conversion, incubations
without addition of glycolaldehyde were carried out. Under our
conditions, without addition of glycolaldehyde, only small
amounts of free glycolaldehyde were detected (Table 1).
Furthermore, incubations in the absence of BSA and TKA
revealed formation of just 3 mol % glycolaldehyde after 24 h
26,27
the production of L-erythrulose.
Glycolaldehyde represents
one of the best nonphosphorylated acceptor substrates for TKs
from nonmammalian organisms (K = 14 mM, glycolaldehyde
M
22
for TKA). Generally, however, phosphorylated substrates
have higher affinities toward TK (K = 1.4 mM, ribose-5-
M
22
phosphate for TKA). Obviously, the addition of glycolalde-
(
data not shown).
Conversion of Sugar Phosphates, Erythrulose, Glyco-
hyde as a competing acceptor substrate led to decreased
formation of sedoheptulose-7-phosphate (82 vs 87 mol %).
This was also envisioned by less depletion of ribose-5-
phosphate in the presence of glycolaldehyde (13.5 vs 11.5
laldehyde, and Glyoxal. Figure 2 demonstrates the
mol %). As the reaction proceeds, both β-hydroxypyruvate (K
M
22
22
=
18 mM) and sedoheptulose-7-phosphate (K = 4 mM)
M
can serve as donor substrates for glycolaldehyde, respectively.
3
,7,8
As posted in the literature,
the theoretical cleavage of the
carbanion DHEThDP intermediate by protonation might
release glycolaldehyde and should entail an immediate
conversion to erythrulose during one-substrate-catalyzed
reactions. However, the release of free glycolaldehyde was
not substantiated so far. To further investigate this hypothesis
in the present setup, BSA should serve as a trap for
glycolaldehyde released from one-substrate reactions, which
would result in elevated amounts of protein modifications.
However, neither the formation of glycolaldehyde nor HEL
significantly increased over time (Tables 1 and 2) in absence of
externally added glycolaldehyde. Thus, the rate of formation of
DHEThDP is considerably higher than its cleavage rate and due
to the very small amounts of the intermediate actually present
during the turnover in the presence of an acceptor aldose,
cleavage of DHEThDP entailing a possible release of
Figure 2. Conversion of ribose-5-phosphate (•) to sedoheptulose-7-
phosphate (■) by transketolase (closed symbols) or inactivated
transketolase (open symbols) in the presence of BSA and
glycolaldehyde.
Table 2. Formation of AGEs in mmol/mol L-Leucine (Mean Concentration ± Standard Deviation) by Transketolase A (TKA)
or Inactivated Transketolase A (ITKA) at 8 h of Incubation Time, with and without addition of 10 mol % Glycolaldehyde (GA)
TKA without GA
ITKA without GA
TKA with GA
ITKA with GA
a
b
HEL
3.4 ± 0.2
0.62 ± 0.05
0.034 ± 0.002
4.1 ± 0.3
6.2 ± 0.2
0.85 ± 0.02
0.049 ± 0.002
18 ± 1
b
CML
0.64 ± 0.03
0.033 ± 0.002
1.6 ± 0.1
a
imidazolinone
0.09 ± 0.01
a
b
Significant difference compared to TK by t-Test (α = 0.05). Significant difference compared to TK by WELCH-Test (α = 0.05).
D
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Journal of Agricultural and Food Chemistry
Article
glycolaldehyde must be evaluated to have only a negligible
impact, if any on the formation of AGEs in vivo.
basically leads to the very same AGEs, it was also monitored in
the present study (Figure 3B). As expected, formation of
glyoxal was up to 4 times higher after addition of
glycolaldehyde (0.31 mol %, inactivated TKA vs 1.17 mol %,
inactivated TKA). In the presence of active TKA, concen-
trations were significantly lowered (Table 1). Since glyoxal is
not metabolized by TKs, these differences can only be
explained by the enzymatic withdrawal of the precursor
compound glycolaldehyde from the incubation system.
2
,14
Nevertheless, it has to be stated that despite the lack of
evidence for the transketolase triggered release of glycolalde-
hyde, incubations without addition of glycolaldehyde indeed
revealed detectable amounts of glycolaldehyde (0.1 mol %,
TKA vs 0.5 mol %, inactivated TKA, 8 h). In the absence of
transketolase activity, this has to be attributed to the
spontaneous and Maillard-induced decomposition of β-
hydroxypyruvate and other sugars. On the other hand, the
significant decrease in the presence of transketolase was
paralleled by an almost quantitative increase of erythrulose
Formation of Protein Modifications. As TKA signifi-
cantly renders concentrations of glycolaldehyde and other
carbonyl compounds, all reactions were performed in the
presence of BSA to explore the impact on the formation of
Maillard protein modifications. The reaction of glycolaldehyde
with the ε-amino function of protein-bound lysine starts with
the formation of the Schiff base adduct followed by
(
0.6 mol %, 8 h), which again underpins the pronounced
acceptor qualities of glycolaldehyde.
Consequently, transketolase led to a substantial formation of
erythrulose in the presence of glycolaldehyde with up to 5 mol
4
,5
%
after 8 h approaching a steady-state afterward (Figure 3A).
rearrangement to the Amadori product. Both forms were
6
converted to N -(2-hydroxyethyl) lysine (HEL) by reduction
with NaBH at the time of sampling, prior to acidic hydrolysis.
4
Figure 4A demonstrates that during incubations with
inactivated TKA, HEL was formed progressively until a plateau
was reached at about 12−24 h with 21 mmol/mol L-leucine. On
Figure 3. Formation of erythrulose (A) and glyoxal (B) in the
presence of BSA and glycolaldehyde by transketolase (closed bars) or
inactivated transketolase (open bars).
This exactly corresponded to the difference in the amounts of
sedoheptulose-7-phosphate formed in presence vs absence of
glycolaldehyde, and verifies the competitive character of
glycolaldehyde and ribose-5-phosphate as acceptor aldoses
leading to erythrulose and sedoheptulose-7-phosphate, respec-
tively. More importantly, it also confirms the conversion of at
least 50% glycolaldehyde after 8 h. Incubations with inactivated
TKA in the presence of glycolaldehyde showed detectable but
not quantifiable amounts of erythrulose, most likely formed via
aldol condensation reactions. It has to be mentioned that the
overall reaction proceeds much more complex as erythrulose
itself represents a substrate of the TK reaction and may also
lead to sedoheptulose-7-phosphate as depicted in Figure 1.
Nonenzymatic Maillard processes easily oxidize glycolalde-
Figure 4. Formation of protein bound HEL (A), CML (B), and
imidazolinone (C) in the presence of BSA and glycolaldehyde by
transketolase (closed bars) or inactivated transketolase (open bars).
28
hyde to glyoxal which is of major relevance in vivo. Because it
E
DOI: 10.1021/acs.jafc.7b03183
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
the other hand, the presence of TKA significantly hampered
formation of HEL with maximum concentrations at 12 h with 7
mmol/mol L-leucine followed by degradation. Strikingly, the
differences correlated with the enzymatic conversion of
glycolaldehyde to the overall more-stable erythrulose. As
already discussed above, the significant difference of HEL
AUTHOR INFORMATION
■
*
5
Corresponding Author
E-mail: marcus.glomb@chemie.uni-halle.de. Fax: ++049-345-
527341).
ORCID
Alexander Klaus: 0000-0001-9289-5742
Notes
The authors declare no competing financial interest.
(3.4 mmol/mol L-leucine TKA vs 4.1 mmol/mol L-leucine,
inactivated TKA) in incubations without addition of glyco-
laldehyde (Table 2) must be attributed to the nonenzymatic
degradation of β-hydroxypyruvate and other sugars to give
glycolaldehyde followed by the enzyme-catalyzed formation of
erythrulose. Nevertheless, the time course for HEL in Figure
ACKNOWLEDGMENTS
■
We thank Prof. Dr. G. Sprenger (University of Stuttgart,
Germany) and Dr. R. Golbik from the Institute for
Biochemistry and Biotechnology/Microbial Biotechnology for
providing the plasmid pGSJ427 encoding TKA. We would also
4A highlights the glycolaldehyde-Schiff base adduct as an
important transient intermediate of this Maillard reaction
cascade with high reactivity.
̈
like to thank Dr. D. Strohl from the Institute of Organic
After Amadori rearrangement to the aldoamine, oxidation,
hydration, and isomerization results in the quantitatively most
relevant formation of the stable advanced Maillard protein
Chemistry, Halle, Germany, for recording NMR spectra and
Dr. A. Frolov from the Leibniz Institute of Plant Biochemistry,
Halle, Germany, for performing accurate mass determination.
Funding was supported by the Deutsche Forschungsgemein-
schaft (DFG, Germany) Research Training Group 2155,
ProMoAge.
6
modification N -carboxymethyl lysine (CML) (Figure 4B). In
incubations with inactivated TKA, levels of CML accumulated
over time, which supports the mechanism and also nicely
correlated to the accumulation of glyoxal (Figure 3B). In the
presence of TKA, the formation was almost stalled leading to a
ABBREVIATIONS USED
■
4
1
:1 ratio (6.0 ± 0.4 mmol/mol L-leucine, inactivated TKA, vs
.37 ± 0.02 mmol/mol L-leucine, TKA) after 24 h. Contrary to
AGEs, advanced glycation endproducts; BSA, bovine serum
7
6
albumin; CMA, N -carboxymethylarginine; CML, N -carbox-
ymethyl lysine; DHEThDP, dihydroxyethyl-thiamine diphos-
phate; dihydroxyimidazolidine, 5-(4,5-dihydroxy-2-imino-1-imi-
dazolidinyl) norvaline; GA-N, 2-(naphthalene-1-ylamino)-
the formation of HEL in incubations without addition of
glycolaldehyde, levels of CML were almost identical whether
with TKA or with inactivated TKA after 8 h (Table 2). These
findings must be explained with the mechanistically more
delayed formation of the stable endproduct.
6
ethanol; HEL, N -(2-hydroxyethyl) lysine; HFBA, heptafluor-
obutyric acid; imidazolinone, 5-(2-imino-5-oxo-1-imidazolidin-
yl) norvaline; ThDP, thiamine diphosphate; TK, transketolase;
TKA, transketolase A
11
As described by Glomb and Lang, 5-(2-imino-5-oxo-1-
imidazolidinyl) norvaline (imidazolinone) is a major arginine
7
modification, arising from the acid-catalyzed conversion of N -
carboxymethylarginine (CMA) and 5-(4,5-dihydroxy-2-imino-
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