Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate-2-Kinase

Article abstract of DOI:10.1002/cbic.201700201

The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by ³¹P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.

Full text of DOI:10.1002/cbic.201700201

Accepted Article
Title: Biocatalytic Asymmetric Phosphorylation catalyzed by
recombinant Glycerate-2-kinase
Authors: Norman Hardt, Birhanu M Kinfu, Jennifer Chow, Wolfgang
R. Streit, Bernhard Schoenenberger, Markus Obkircher, and
Roland Wohlgemuth
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To be cited as: ChemBioChem 10.1002/cbic.201700201
Link to VoR: http://dx.doi.org/10.1002/cbic.201700201
A Journal of
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10.1002/cbic.201700201
ChemBioChem
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Biocatalytic Asymmetric Phosphorylation catalyzed by
recombinant Glycerate-2-kinase
Norman Hardt[a], Birhanu M. Kinfu[b], Jennifer Chow[b], Bernhard Schoenenberger[a],Wolfgang R. Streit*[b],
Markus Obkircher[a] and Roland Wohlgemuth*[a]
Abstract: The efficient synthesis of pure
D
-Glycerate-2-phosphate is
characterized as a crystalline
salt by Meyerhof and Kiessling.[1] An efficient, robust and
scalable route for the preparation of a soluble form of pure
D-glycerate-2-phosphate barium
of much interest due to its importance as enzyme substrate and
metabolite. A straightforward one-step biocatalytic phosphorylation
of glyceric acid has been investigated. Glycerate 2-kinase from
Thermotoga maritima has been expressed in Escherichia coli allow-
ing easy purification. The selective glycerate 2-kinase-catalyzed
phosphorylation has been followed by 31P-NMR and showed ex-
D
-
glycerate-2-phosphate has been of much interest since its
discovery. It continues to be required both as a metabolite
reference standard as well as an enzyme substrate for mea-
suring activities of enolases and their inhibitors, e.g. enolase
activities in a number of human diseases and disease-causing
human pathogens such as Streptococcus mutans and the
inhibiting action of fluoride or phosphate in caries prevention,
Staphylococcus aureus, Streptococcus pyogenes or the blood
parasites Schistosoma mansoni causing schistosomiasis and
Trypanosoma brucei causing sleeping sickness.[2] The first
cellent enantioselectivity towards the phosphorylation of the
D-
enantiomer of glyceric acid. This straightforward phosphorylation
reaction and the subsequent product isolation has enabled the
preparation of enantiomerically pure
phosphorylation reaction using recombinant glycerate 2-kinase
yields -Glycerate 2-phosphate in less reaction steps and with
D-Glycerate 2-phosphate. This
D
higher purity than chemical routes.
definitive synthesis of
described by Ballou and Fischer.[3] The identity and purity of the
final product -glycerate-2-phosphate, which was obtained as
D-glycerate-2-phosphate has been
D
the crystalline trisodium salt pentahydrate, was proven, but nine
reaction steps were required to get from the starting compound
Introduction
D
-galactose to the final product. Although a shorter synthesis
-gluconolactone has been described,[4] the
D
-Glyceric acid 2-phosphate (scheme 1) is a key metabolite
starting from
D
in central carbon metabolism, glycolysis/gluconeogenesis,
pentose phosphate pathway, glycine, serine and threonine
metabolism, methane metabolism, biosynthesis of plant
secondary metabolites, phenylpropanoids, terpenoids, steroids,
alkaloids derived from the shikimate pathway, antibiotics and
amino acids.
bottlenecks in chemical synthesis and the number of reaction
steps using protection-deprotection and purification steps have
created interest in an attractive direct enzymatic phosphorylation
of glyceric acid.[5] The enzymatic phosphorylation of glyceric acid
is also of clinical relevance for patients with D-glyceric aciduria, a
rare inborn error of serine and fructose metabolism, as
mutations in the GLYCTK gene have been revealed as the
cause of D-glycerate kinase deficiency and D
-glyceric aciduria.[6]
Site- and stereoselective kinases have been very powerful
and versatile catalysts for biocatalytic phosphorylations of small
molecules, e.g. in straightforward syntheses of phosphorylated
metabolites.[7] As class II glycerate kinases catalyze the
formation of D-glyceric acid 2-phosphate in nature, a stable and
highly selective glycerate kinase from this class (EC 2.7.1.165)
has been selected for the biocatalytic phosphorylation of
glycerate in the 2-position.
Scheme 1. D-Glyceric acid 2-phosphate
The glycolytic intermediate between
D-glycerate-3-phosphate
and phosphoenolpyruvate was first isolated from yeast and
[a]
[b]
Dr.N.Hardt, Dr.B.Schoenenberger, Dr.M.Obkircher,
Dr.R.Wohlgemuth*
Sigma-Aldrich, Member of Merck Group, Industriestrasse 25,
CH-9470 Buchs, Switzerland
E-mail: roland.wohlgemuth@sial.com
B.M.Kinfu, Dr.J.Chow, Prof. W.R.Streit*
Universität Hamburg, Abteilung für Mikrobiologie und
Biotechnologie, Ohnhorststrasse 18, 22609 Hamburg, Germany
E-mail: wolfgang.streit@uni-hamburg.de
Scheme 2. Biocatalytic phosphorylation of
D-glyceric acid by glycerate 2-
kinase using the phosphoenolpyruvate(PEP)/pyruvate kinase-system for the
regeneration of ATP
Supporting information for this article is given via a link at the end of
the document.
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While a number of glycerate kinases from rat liver,[8]
Enantioselectivity of the glycerate 2-kinase-catalyzed
phosphorylation of glycerate
Escherichia coli,[9] Pyrococcus horikoshii,[10] Picrophilus torridus,
Thermoplasma acidophilum,[12] Sulfolobus tokodaii[13] and
[11]
The enantioselectivity was determined by analyzing the kinetics
of the glycerate 2-kinase-catalyzed conversion of equal amounts
Hyphomicrobium methylovorum,[14] which are able to catalyze
the formation of glycerate-2-phosphate, were investigated,
detailed enzymatic information on the key enzyme glycerate 2-
kinase was reported on the glycerate kinase of the hyper-
thermophilic archaeon Thermoproteus tenax.[15] From the com-
parison of the kinetic and biochemical properties of the characte-
rized class II glycerate kinases of T. tenax, P. torridus, T. acido-
philum and H. methylovorum, the glycerate 2-kinase from
Thermotoga maritima was selected due to its favorable bioca-
talytic properties and structure-function characterization.[16-17]
The 1.25 kb glycerate-2-kinase gene from Thermotoga
maritima was expressed heterologously in E. coli as a fusion
protein with the maltose-binding-protein (MBP) and was purified
by affinity chromatography. The active form of the protein was
obtained by cleaving off the MBP. We have investigated biocata-
lytic phosphorylations catalyzed by recombinant glycerate-2-ki-
nase with quantitative 31P-NMR spectroscopy using phospho-
enolpyruvate/pyruvatekinase-system for ATP-regeneration, star-
of DL-glyceric acid, D-glyceric acid and L-glyceric acid. Every 15
minutes a 1H-NMR spectrum (left) and a 31P-NMR spectrum
(right) has been measured. T=0 (red, bottom) is measured app.
2 minutes after addition of the enzymes. The conversion can be
tracked by the decrease of the PEP signals (1H: 5.0-5.4 ppm;
31P: -0.9 ppm) and the increase of the product signals of
D-
glyceric acid 2-phosphate and pyruvate. (1H: 4.4 ppm and 2.3
ppm; 31P: 2.4 ppm), as shown in figures 2 and 3.
A)
ting with racemic and the enantiopure
D
- and
L-glycerate as
substrate. Nearly 100% conversion of
te-2-phosphate was found.
D
-glycerate to D
-glycera-
With pure
L-glycerate as substrate, no
L-glycerate-2-
phosphate was formed, while DL-glycerate gave 50% conversion
with excellent enantioselectivity.
Results and Discussion
The glycerate-2-kinase gene from Thermotoga maritima was
cloned into pMAL-C2X vector and expressed in E. coli as
soluble fusion protein with MBP (87 kDa). Highest protein yield
was obtained when expressed at a temperature of 17 °C. After
harvesting and purification using affinity chromatography with
amylose resins, the active glycerate 2-kinase (52 kDa) has been
obtained by cleaving off the MBP with factor Xa protease
maintaining its maximum activity without any tag.
Figure 2. (A) NMR-Kinetics of the enzymatic conversion of DL-Glyceric acid.
10 mg DL-Glyceric acid (94 µmol, 1.0 eq), 2.6 mg ATP (5 µmol, 0.05 eq) and
17.5 mg PEP (85 µmol, 0.9 eq) were dissolved in 1.7 ml D2O with 10mM
MgCl2. pH was adjusted to 7. Subsequently 50 Units pyruvate kinase (in 50 µl)
and 250 µl of the prepared recombinant glycerate 2-kinase (Thermotoga
maritima) were added. The enzymatic reaction was observed via NMR. (B)
NMR-Kinetics of enzymatic conversion of
L-Glyceric acid, whereby the same
procedure as in (A) was followed except that instead of DL-Glyceric acid as
substrate 10 mg L-Glyceric acid (94 µmol, 1.0 eq) was used.
B)
Figure 1. SDS-PAGE analysis of expression and purification of glycerate 2-
kinase. Wash and elution fraction of purified fusion protein with MBP (A) and
cleaved protein after using factor Xa protease (B). Approximately 1.5 (A) and
35 µg (B) total proteins were loaded, respectively. M: marker, W: wash, E:
eluate, C: cleaved protein.
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As shown in figure 4, glycerate 2-kinase from Thermotoga
maritima is most active at neutral and alkaline pH and its
maximum activity was recorded at pH 9.2. The enzyme also
maintained its full activity after storage at 4°C for several months
with 0.02% NaN3 (w/v). Its excellent stability (both reaction and
storage) as well as activity profile, thus allowing a direct coupling
to the ATP regeneration system for glycerate 2-phosphate
synthesis, is an advantage of this glycerate 2-kinase from
Thermotoga maritima.
An optimized reaction system for the biocatalytic phosphoryla-
tion of D-glyceric acid acid is however not only dependent on the
optimum properties of the glycerate 2-kinase, but also on the
optimum parameters for ATP-regeneration and the stability
profile of
acid 2-phosphate has been examined in the pH range of 6.0 to
11.0. A 20 mM solution of the lithium salt of -glyceric acid 2-
D-glyceric acid 2-phosphate. The stability of D-glyceric
D
phosphate in citrate buffer at pH 6.0, in Tris buffer at pH 7.0 and
8.0 as well as in carbonate buffer at pH 9.0, 10.0 and 11.0 was
1
observed by H- and 31P-NMR. No decomposition was detected
Figure 3. NMR-Kinetics of enzymatic conversion of
D
-Glyceric acid. The same
during the time period of observation, which was 5 days for pH
10.0 and 11.0, 11 days for pH 7.0, 8.0 and 9.0 and 18 days for
pH 6.0. As the pH optimum of the ATP regeneration system is at
procedure as in figure 2 was applied, but 10 mg
eq) was used as starting material.
D-Glyceric acid (94 µmol, 1.0
pH 7.0 to 7.5, the biocatalytic phosphorylation of D-glyceric acid
The NMR-experiments in figures 2 and 3 clearly demonstrate
the conversion of only the -glyceric acid and no conversion of
the -glyceric acid to the corresponding product phosphorylated
in the 2-position.
at a pH around 7 is favoured over a reaction pH at the optimum
pH of the glycerate 2-kinase, although the stability profile of the
D
L
D
-glyceric acid 2-phosphate would be compatible.
In order to determine the enantiomeric purity of the product,
capillary zone electrophoresis using vancomycin as chiral
selector was performed and confirmed an enantiomeric ratio of
Kinetic measurements of glycerate 2-kinase from
Thermotoga maritima
98.3:1.7=D:L for the product D-glycerate 2-phosphate (see direct
data on capillary zone electrophoresis in the supporting
information). The development of suitable reaction conditions for
complete conversion in phosphorylations is facilitated by 31P-
NMR reaction monitoring,[18] as both product formation as well as
phosphoenolpyruvate donor consumption can be measured
simultaneously. From the time course of biocatalytic O-
A Km value of (0.13 +/- 0.03) mM to D-glyceric acid, a catalytic
turnover number kcat of (5.4 +/- 0.7) 10-2 s-1 and a kcat/Km of (4.0
+/- 0.05) 104 M-1s-1 has been determined at 37°C for this glyce-
rate 2-kinase (see experimental section). The Km value for our
glycerate 2-kinase agrees well with the wild-type TM1585. While
the kcat for the D-glycerate was not determined in the wild-type
phosphorylation of D-glyceric acid in the 2-position, followed by
31P-NMR as shown in figure 5, the reaction conditions for
TM1585, the temperature dependence of the specific activity
showed a large increase from 37°C to 80°C, levelling off at
90°C.17
complete conversion and optimal process design can be derived.
4.00
3.00
100
80
60
40
20
0
2.00
2.9 ppm
-0.94 ppm
1.00
0.00
0
50
100
150
200
Time/ min
-1.00
2
3
4
5
6
7
8
9
10
11
pH
Figure 5. Kinetics of formation of D-glycerate-2-phosphate (blue curve) and
consumption of phosphoenolpyruvate (red curve) derived from the 31P-NMR
signals.
Figure 4. Glycerate 2-kinase activity as a function of pH. 50 mM citrate buffer
pH 3.0, 4.0 5.2 and 6.2 ( ), 50 mM Tris-HCl buffer pH 7.4 and 8.0 ( ) and
50 mM sodium carbonate-bicarbonate buffer pH 9.2 and 10.2 ( ).
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of enzymatic phosphorylations of DL-glyceric acid, L-glyceric acid and D-
Conclusions
1
glyceric acid was measured by the acquisition each 15 minutes of a H-
NMR (left) and a 31P-NMR (right). T=0 (red, bottom) is measured app. 2
minutes after addition of the enzymes. Conversion was tracked by the
decrease of the PEP signals (1H: 5.0-5.4 ppm; 31P: -0.9 ppm) and the
increase of product signals (1H: 4.4 ppm; 31P: 2.4 ppm).
Glycerate 2-kinase from Thermotoga maritima was selected as
biocatalyst for the phosphorylation of glyceric acid and the
corresponding gene was expressed in E.coli as fusion protein
with MBP to allow easy affinity purification. After cleaving off
MBP by factor Xa protease a highly active and stable glycerate
2-kinase has been obtained. A Km value of (0.13 +/- 0.03) mM to
glyceric acid, a catalytic turnover number kcat of (5.4 +/- 0.7) 10-2
Capillary Electrophoresis
Capillary zone electrophoresis with EOF-reversal (dynamic coating) was
performed with a PDA-detector and indirect detection at a wavelength of
233 nm and at 25 C. Noncoated capillaries with 75 µm inner diameter,
120 cm length and 110 cm to the detector were used. Accelerator from
CEofix anions 5 Kit was used as electrophoresis buffer, initiator from
CEofix anions 5 Kit as conditioner and 40 mM Vancomycin-solution as
chiral selector.
s
-1 and a kcat/Km of (4.0 +/- 0.05) 104 M-1s-1 has been determined.
The glycerate 2-kinase-catalyzed phosphorylation of the - and
-enantiomer of glyceric acid as well as its racemic form have
D
L
been directly investigated by NMR. The experiments clearly
demonstrated the power and selectivity of biocatalytic
phosphorylation. Only the
D-glyceric acid was accepted as a
substrate for this recombinant glycerate 2-kinase, while the
L
-
Amplification and cloning of glycerate 2-kinase gene
glyceric acid was not phosphorylated at all. This straightforward
and highly selective phosphorylation reaction is therefore very
valuable for the enzymatic resolution of racemic glyceric acid or
Glycerate kinase gene from T. maritima was amplified with PCR using
primer pairs G2K-EcoRI_F (5’ - gaattcATGTTTGATCCTGAATCCTTG
and G2K-HindIII_R (5’ - aagcttCTAGACGATGAGGCCTATTATC) and
cloned into pDrive cloning vector. The gene was then excised out from
the plasmid with endonucleases EcoRI and HindIII and subcloned into
pMAL-C2X in the same translational reading frame as the malE gene for
for the synthesis of
of -glyceric acid in the 2-position.
The glycerate 2-kinase-catalyzed phosphorylation of
acid and the subsequent product isolation has enabled the
preparation of the key metabolite -glycerate 2-phosphate in
D-glycerate 2-phosphate by phosphorylation
D
D
-glyceric
a
N-terminal MBP-fused expression. The clone was verified via
D
sequencing using primers mal2E (5’ - AGCTGGCAAAAGAGTTCC) and
M13 forward (5’ – TGTAAAACGACGGCCAGTG) both binding within
vector sequence upstream (within malE gene) and downstream of the
insert. The fusion plasmid clone was finally transformed using heat-shock
into chemically competent Escherichia coli BL21 (DE3) host cells for
expression.
enantiomerically pure form. This biocatalytic phosphorylation
reaction using recombinant glycerate 2-kinase is also very
sustainable, as it yields
D-glycerate 2-phosphate in a one-step
reaction and with higher purity than chemical routes.
The excellent enantioselectivity and the first crystal structure[16]
of a representative of the glycerate 2-kinase family provide
exciting opportunities for extending this work to new phospho-
rylated compounds which up to now are only accessible though
Culture conditions, protein expressions and purification
lengthy chemical routes, such as substituted D-glycerate analo-
E. coli BL21 (DE3) expression host cells carrying the right fusion clone
were grown overnight at 37 °C in rich medium (10 g tryptone, 5 g yeast
extract and 5 g NaCl) with 2% glucose and 100 µg/ml Ampicillin. Three
per cent of the overnight grown cells were transferred into same fresh
medium; further incubated until OD600 is 0.6 and induced with 0.3 mM
gues and functional isosteric and negatively charged substrates
able to dock properly into the active site. Although highly
conserved amino acid residues from the Rossmann-like domain
and the C-terminal domain have been identified and contain one
highly conserved basic residue, a detailed understanding of the
isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 17 °C for 18 hrs for
maximum soluble protein expression. Cells were harvested using
centrifugation and resuspended in column buffer (20 mM Tris-Cl pH 7.4,
0.2 Mm NaCl, 1 mM EDTA, 20 mM 2-ME and 1 mM NaN3). French press
was used to breakup cells and the supernatant after centrifugation was
loaded onto pre-equilibrated column packed with amylose resins. After
washing with 12 volume of column buffer, the fusion protein was eluted
with 10 mM maltose in same buffer.
interaction with the negatively charged
D-glycerate and the
elucidation of the mechanism of the enzyme function require
further studies and higher resolution.
Experimental Section
In order to get tag-free glycerate 2-kinase protein, the MBP was cleaved
off from the fusion protein with Factor Xa protease (New England
Biolabs) overnight on ice. The protein was further concentrated and
simultaneously dialyzed using Vivaspin columns (Sartorius) and then
quantified with Roti®-Quant solution (Carl Roth) according to
manufacturer’s protocol.
Unless otherwise stated, all chemicals were of analytical grade and were
purchased from Sigma-Aldrich.
Kinetic measurements of glycerate 2-kinase from Thermotoga
NMR spectroscopy
maritima
NMR-spectra were measured in D2O at room temperature on a Bruker
Avance III 600 MHz spectrometer equipped with a BBO probe head with
z-gradient using 600.2 MHz for 1H, 150.9 MHz for 13C. The NMR-kinetics
Kinetic determinations of the glycerate 2-kinase were performed using a
continuous coupled assay based on the oxidation of NADH, where the
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10.1002/cbic.201700201
ChemBioChem
FULL PAPER
decrease in absorbance at 340 nm was monitored using a SynergyTM HT
multi-mode microplate reader with Gen5 software. The assay mixture
contained 0.3 mM NADH, 1.5 mM ATP, 10 mM MgSO4, 1.5 mM
Acknowledgements
We would like to thank Dr. Eduard Frick for the capillary electro-
phoresis analysis and the German Federal Ministry of Education
and Research (BMBF) for the support of project P28 under the
cluster of Biocatalysis2021
phosphoenolpyruvate, 1.5 U of pyruvate kinase, 1.5 U of
L-lactate
dehydrogenase and 0.1 to 1.4 mM varying D-glyceric acid concenatrions
varying from 0.1 to 1.4 mM in 50 mM Tris buffer pH 7.4 to a volume of
200 µl. The reactions were carried out in triplicates at 37°C after adding
0.2 µg purified glycerate 2-kinase. Apparent values of kcat and Km were
determined applying the Michaelis-Menten kinetic model.
A
Keywords: D-Glycerate-2-phosphate • Glycerate-2-kinase •
discontinuous assay was however used for measuring the enzyme
activity over varying pH conditions from pH 3.0 to 10.2. The
phosphorylation reaction took place separately at 37°C for 10 minutes
and put immediately on ice. The pH of assay reactions was adjusted by
adding predetermined volumes of NaOH or HCl just before coupling to
the indicator reaction which had 5 times excess pyruvate kinase and
lactate dehydrogenase. The ADP formed was then measured by
monitoring oxidation of NADH same as above.
asymmetric biocatalysis • enzymatic phosphorylation • chiral
metabolite
[1] O.Meyerhof, W.Kiessling, Biochem. Z. 1935, 276, 239-
[2] a) V.Pancholi, Cellular and Molecular Life Sciences CMLS 2001 58, 902-
920; b) M.Kaufmann, P.Bartholmes, Caries Research 1992, 26, 110-116; c)
Y.Wu, C.Wang, S.Lin, M.Wu, L.Han, C.Tian, X.Zhang, J.Zang, Acta Crystallo-
gr. D Biol. Crystallogr. 2015, 71, 2457-2470; d) J.A.Kornblatt, V. Quiros,
M.J.Kornblatt, PLoS ONE 2015, 10, e035754; e) B.C.Figueiredo, A.A.Da'dara,
S.C.Oliveira, P.J.Skelly, PLoS Pathog. 2015, 11 , e1005335; f) V. Hannaert,
M.-A.Albert, D.J.Rigden, M.T.da Silva Giotto, O.Thiemann, R.C. Garratt, J.Van
Roy, F.R.Opperdoes, P.A.M.Michels, Eur. J. Biochem. 2003 270, 3205–3213.
[3] C.E.Ballou, H.O.L.Fischer, J. Am. Chem. Soc. 1954, 76, 3188-3193.
[4] F.C.Hartman, F.Wold, Biochimica et Biophysica Acta (BBA)-General
Subjects, 1967, 141, 445-453.
Enzyme reactions
Enzymatic syntheses of DL-,
mg of respectively DL-, - and
D
- and
L-Glyceric acid 2-phosphate. 10
D
L-Glyceric acid (94 µmol, 1.0 eq), 2.6 mg
ATP (5 µmol, 0.05 eq) and 17.5 mg PEP (85 µmol, 0.9 eq) were
dissolved in 1.7 ml D2O with 10mM MgCl2. pH was adjusted to 7.
Subsequently 50 Units pyruvate kinase (in 50 µl) and 250 µl of the
recombinant glycerate 2-kinase were added. The enzymatic reaction was
observed via NMR. 1H-NMR and 31P-NMR were measured every 15 min.
[5] P.A.Sims, G.H.Reed, J. Mol. Catal. B: Enzym 2005, 3, 77-81.
[6] J.O.Sass, K.Fischer, R.Wang, E.Christensen, S.Scholl-Bürgi, R.Chang,
K.Kapelari, M.Walter, Hum. Mutat., 2010, 31, 1280–1285.
[7] a) R.Wohlgemuth, A.Liese, W.Streit, Trends Biotechnol. 2017, http://dx.doi.
org/10.1016/j.tibtech.217.01.005, in press; b) D.Gauss, B.Schönenberger, G.S.
Molla, B.M.Kinfu, J.Chow, A.Liese, W.Streit, R.Wohlgemuth, in: Applied
Gram-scale preparation of
D-glycerate-2-phosphate catalyzed by
recombinant glycerate 2-kinase
Biocatalysis
-
From Fundamental Science to Industrial Applications
(Eds.:A.Liese, L.Hilterhaus, U.Kettling, G. Antranikian) ISBN: 978-3-527-
33669-2, Wiley-VCH, Weinheim, Germany, 2016, 147-177.
[8] W.Lamprecht, F.Heinz, T.Diamartstein, Zeitschrift Physiolog. Chem. 1962,
328, 204-206.
1.0 g D-Glyceric acid (9.4 mmol, 1.0 eq), 260 mg ATP (0.5 mmol, 0.05
eq) and 1.75 g PEP (8.5 mmol, 0.9 eq) were dissolved in 200 ml 10mM
MgCl2 solution. The pH value was adjusted to 7 and subsequently 1000
Units pyruvate kinase (in 1000 µl) and 1100 µl glycerate 2-kinase
(Thermotoga maritima) were added and stirred gently. The conversion of
the enzymatic reaction was observed by NMR and reached completion
after 24h. Subsequently the enzyme was filtered off using a 10000 MW
[9] L.Zelcbuch, M.Razo-Mejia, E.Herz, S.Yahav, N.Antonovsky, H.Kroytoro,
R.Milo, A.Bar-Even, PLoS ONE 2015, 10, e0122957.
[10] B.Liu, Y.Hong, L.Wu, Z.Li, J.Ni, D.Sheng, Y.Shen, Extremophiles 2007,
11, 733-739.
Zentricon filter membrane. To the resulting reaction solution containing
glycerate 2-phosphate 3.3 g calcium acetate monohydrate (28.2 mmol, 3
eq) was added to precipitate the -glycerate 2-phosphate as its calcium
D-
[11] a) M.Reher, M.Bott, P.Schönheit, FEMS Microbiol. Lett. 2006, 259, 113-
119; b) M.Reher, P.Schönheit, FEBS Lett. 2006, 580, 1198-1204.
[12] M.Noh, J.H.Jung, S.B.Lee, Biotech. Bioprocess Eng. 2006, 11, 344-350.
[13] B.Liu, L.Wu, T.Liu, Y.Hong, Y.Shen, J.Ni, Biotechnol. Lett. 2009, 31,
1937-1941.
D
salt and harvest it by filtration. This calcium salt was finally converted
into the corresponding lithium salt using 150 ml lithium conditioned
Dowex 50W X8 H+ ion exchange resin. The solution of the D-glycerate 2-
[14] T.Yoshida, K.Fukuta, T.Mitsunaga, H.Yamada, Y.Izumi, Eur. J. Biochem.
1992, 210, 849-854.
phosphate lithium salt was evaporated to give 1.255 g white solid product
in 72% yield and excellent purity.
[15] D.Kehrer, H.Ahmed, H.Brinkmann, B.Siebers, BMC Genomics 2007, 8,
301.
1H NMR (D2O, 600 MHz): δ 4.48 (ddd, J = 8.6, 5.4, 3.1 Hz, 1H), 3.89 (dd,
J = 11.9, 3.1, 1H), 3.82 (dd, J = 11.9, 5.4 Hz, 1H).
13C NMR (D2O, 151 MHz): δ 177.41 (d, J = 5.6 Hz), 76.66 (d, J = 5.5 Hz),
64.24 (d, J = 3.6 Hz).
31P NMR (D2O, 162 MHz): δ 2.15 (s, 1P).
MS: found: 185.0; calculated: 185.0 (M-H+, C3H7O7P-);
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Koesema, J.S.Kovarik, A.Kreusch, P.Kuhn, T.M.McPhillips, A.T.Morse, K.
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2008, 190, 1773-1782.
Capillary Electrophoresis: Enantiomeric Ratio D:L = 98.3:1.7
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10.1002/cbic.201700201
ChemBioChem
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Recombinant glycerate 2-kinase from
Thermotoga maritima showed an
excellent selectivity towards the phos-
phorylation of the D-enantiomer of
glyceric acid, while the L-enantiomer
was not phosphorylated at all. This
straightforward phosphorylation reac-
tion and the subsequent product isola-
tion has enabled the preparation of
the central glycolytic metabolite D-gly-
cerate 2-phosphate in less reaction
steps and with higher purity than
chemical routes.
Norman Hardt, Birhanu M. Kinfu,
Jennifer Chow, Bernhard
Schoenenberger, Wolfgang R. Streit,
Markus Obkircher and Roland
Wohlgemuth*
Page No. – Page No.
Biocatalytic Asymmetric
Phosphorylation catalyzed by
recombinant Glycerate-2-kinase
This article is protected by copyright. All rights reserved.