Phosphorylation Catalyzed by Dihydroxyacetone Kinase

Article abstract of DOI:10.1002/ejoc.201800350

Site- and enantioselective kinases have been very useful catalysts for biocatalytic phosphorylations in straightforward syntheses of phosphorylated metabolites. Biocatalytic phosphorylations catalyzed by recombinant dihydroxyacetone kinase beyond the dihydroxyacetone substrate have been investigated with quantitative ³¹P NMR spectroscopy using pyruvate-kinase-catalyzed ATP regeneration. A nearly 100 % conversion of d-glyceraldehyde to d-glyceraldehyde 3-phosphate has been found. Interestingly, with pure l-glyceraldehyde as substrate, practically no formation of l-glyceraldehyde 3-phosphate was observed.

Full text of DOI:10.1002/ejoc.201800350

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Title: Dihydroxyacetone kinase-catalyzed Phosphorylation
Authors: Dominik Gauss, Israel Sánchez-Moreno, Isabel Oroz-Guinea,
Eduardo García-Junceda, and Roland Wohlgemuth
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Dihydroxyacetone kinase-catalyzed Phosphorylation
Dominik Gauss,[a] Israel Sánchez-Moreno,[b] Isabel Oroz-Guinea,[b] Eduardo García-Junceda,[b] and
Roland Wohlgemuth*[a]
Abstract: Site- and enantioselective kinases have been very useful
catalysts for biocatalytic phosphorylations in straightforward synthe-
ses of phosphorylated metabolites. Biocatalytic phosphorylations
catalyzed by recombinant dihydroxyacetone-kinase beyond the
dihydroxyacetone substrate have been investigated with quantitative
31P-NMR spectroscopy using pyruvate-kinase-catalyzed ATP-
regeneration. A nearly 100% conversion of D-glyceraldehyde to D-
glyceraldehyde 3-phosphate has been found. Interestingly, with pure
L-glyceraldehyde as substrate, practically no formation of L-
glyceraldehyde 3-phosphate was observed.
biphosphate aldolase/FSA aldolase [5].Therefore we looked for
suitable kinase candidates. Among the different kinases ATP-
dependent dihydroxyacetone kinases are of interest because
they are able to differentiate between hydroxy- and keto-groups.
Dihydroxyacetone kinases (DHAK) are highly conserved and
specific enzymes of microbial glycerol utilization pathways are
occurring widely in plants, animals and some microorganisms. In
an artificial pathway they catalyze the detoxification of
dihydroxyacetone by phosphorylation [6] to the glycolytic
intermediate dihydroxyacetone phosphate [7]. The roles of the
active site residues of dihydroxyacetone kinases have been
investigated by structural and mechanistic studies [8].
Catalytic asymmetric phosphorylations without the use of
protecting groups for the phosphoryl group donors as well as for
the substrates and the development of suitable catalysts are of
major synthetic interest for the introduction of phosphoryl groups
in a selective and sustainable way. As a large number of
catalytic phosphorylation reactions play key roles in biological
cells and are performed with excellent selectivity, it is no
surprise that enzymes have also proven to be very suitable and
versatile catalysts for asymmetric phosphorylation reactions
from laboratory to industrial large-scale [1]. In contrast to chiral
small molecular weight organocatalysts, where the two
enantiomers can be used to catalyze the formation of the two
enantiomeric products, the search for enantiocomplementary
biocatalysts requires various different strategies [2]. For certain
enzyme classes like hydrolases, alcohol dehydrogenases or
transaminases both enantioselective enzymes have been found.
Glycerol kinases have all led to products with the L-configuration
and glycerol kinases with opposite enantioselectivity have so far
not been discovered. Our aim was to extend the kinase-
catalyzed asymmetric synthesis of L-glyceraldehyde 3-
phosphate (L-GAP) [3], which is toxic to cells, to the other
enantiomer and major metabolite D-glyceraldehyde 3-phosphate
(D-GAP). D-GAP was so far obtained by chemical synthesis or a
one-pot enzymatic reaction sequence using the three enzymes
fructose-bisphosphate aldolase, sn-glycerol 3-phosphate
dehydrogenase and formate dehydrogenase [3]. In addition, in
situ D-GAP synthesis has also been performed in different
enzyme cascade reactions, such as acid phosphatase/DERA
aldolases [4] and triosephosphate dehydrogenase/fructose-1,6-
A number of dihydroxyacetone kinases have been applied in the
straightforward preparation of dihydroxyacetone phosphate by
catalyzing the phosphorylation of dihydroxyacetone (Figure 1)
and using ATP as phosphoryl donor [9]. ATP regeneration in
large scale reactions has been successfully solved by using two
different cofactor recycling systems: (i) the enzyme acetate
kinase (AK) with acetyl phosphate as final donor [10] and (ii) the
enzyme pyruvate kinase and the substrate phosphoenol
pyruvate [5].
Figure 1. Dihydroxyacetone kinase-catalyzed phosphorylations of dihydroxy-
acetone and D-glyceraldehyde using ATP as phosphoryl group donor and an
enzymatic regeneration system for the ATP cofactor
The dha operon expression is not only induced by dihydroxy-
acetone, but also by glyceraldehyde [11], which also fits into the
active site of the ATP-dependent dihydroxyacetone kinase. As
several ATP-dependent dihydroxyacetone kinases have been
found to take racemic glyceraldehyde as substrate [12] and
enantiomerically pure D- and L-glyceraldehyde became acces-
sible [13], we investigated racemic glyceraldehyde as well as the
enantiomerically pure D- and L-glyceraldehyde as substrates of
the recombinant Citrobacter freundii dihydroxyacetone kinase
(Figure 1), which was expressed in E. coli.
[a]
[b]
[‡]
Dr. D.Gauss, Dr. R.Wohlgemuth (Corresponding Author)
Sigma-Aldrich, Member of Merck Group
Industriestrasse 25, CH-9470 Buchs, Switzerland
E-mail: roland.wohlgemuth@sial.com
Dr. I.Sanchez-Moreno,[‡] Dr. I.Oroz-Guinea,[‡‡] Dr.E.Garcia-Junceda
Departamento de Química Bioorgánica,
Instituto de Química Orgánica General, CSIC (IQOG-CSIC),
Madrid 28006, Spain
Venter Pharma S.L. Azalea 1, 28109 Alcobendas (Madrid)
[‡‡] Institute of Biochemistry, Dept. of Biotechnology & Enzyme
Using a coupled enzymatic assay for measuring the DHAK
activity (see experimental section) a value of 1.71 U/ml was
obtained for the racemic glyceraldehyde, while with pure D-
Catalysis. Felix-Hausdorff-Str. 4. Greifswald D-17487 Germany
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10.1002/ejoc.201800350
European Journal of Organic Chemistry
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glyceraldehyde a value of 1.93 U/ml was obtained under the
same conditions. However, when L-glyceraldehyde was used as
substrate, no activity was detected even upon increasing
enzyme concentration significantly. In addition, the DHAK
activity was studied as a function of increasing concentrations of
racemic and D-glyceraldehyde and was found to level off at
millimolar concentrations (Figure 2).
Although chromatographic and enzymatic methods have been
very useful for the analysis and development of new biocatalytic
asymmetric phosphorylations,
quantitative
spectroscopic
methods with rich information content like 31P-NMR have greatly
facilitated direct kinetic analyses of such reactions [14].
Therefore we have investigated the same phosphorylation
reactions also by 31P-NMR, which confirmed the enzymatic
assays. While no phosphorylation reaction could be observed
using enantiomerically pure L-glyceraldehyde as substrate, a
nearly 100% formation of D-GAP has taken place after about
two hours reaction time.
Figure 2: DHAK activity at increasing concentrations of (A) D-glyceraldehyde
and (B) D,L-glyceraldehyde. The insert shows the Hanes-Woolf plot.
The KM of 0.15 mM for the racemic D,L-glyceraldehyde was
almost two times than the one for the enantiopure D-
glyceraldehyde (0.072 mM), while the kcat values were almost
coincident (see table below). These results are coherent with the
fact that only the D-glyceraldehyde in the racemic mixture is
being phosphorylated by the DHAK. From these experiments we
can conclude that DHAK is completely stereoselective for D-
glyceraldehyde and that L-glyceraldehyde did not interfere
significantly with the phosphorylation reaction.
Vmax
KM
(mM)
kcat
kcat/KM
Subs
(U·mg-1)
(s-1)
(M-1·s-1)
D,L-GA
D-GA
1.48 ±0.01
0.15 ±0.01
3.13 ±0.03
2.65 ±0.06
20870 ±1405
36805 ±2690
1.25 ±0.03 0.072 ±0.005
Figure 4: 31P-NMR kinetics of dihydroxyacetone kinase-catalyzed phospho-
rylation of D-glyceraldehyde to D-glyceraldehyde 3-phosphate (below) and the
analogous experiment by replacing D-glyceraldehyde by L-glyceraldehyde
(above). Almost no conversion is observed using L-glyceraldehyde as
substrate, whereas D-glyceraldehyde shows an almost 100% conversion after
2h. As D-glyceraldehyde 3-phosphate is not stable under neutral conditions
[3], a decrease of the formed product and a simultaneous increase of newly
formed inorganic phosphate (Pi) is observed.
Stereoselective phosphorylation can be used for the kinetic
resolution of racemic glyceraldehyde. Experiments with racemic
glyceraldehyde have shown a conversion to the 3-phosphoryla-
ted compound corresponding to about 50% of the amount
observed with the pure D-enantiomer, thus resolving racemic
DL-glyceraldehyde by phosphorylation (figure 3).
Due to the low stability of D-GAP under neutral pH conditions
[3], the phosphorylated glyceraldehyde product formed decom-
poses slowly, accompanied by the formation of inorganic phos-
phate (figure 4). This degradation can be controlled by using an
adequate buffer and reaction pH, as previously reported [5] or by
keeping a short reaction time and a rapid change to pH 4 after
ending the reaction [3]. Since the D-GAP purification by
precipitation as a Ba2+ salt intermediate requires acidic pH, it
does not contribute to the D-GAP degradation and the Ba2+ salt
intermediate is stabilized for further processing [3,5], which is
being considered and optimized.
Figure 3. Resolution of DL-glyceraldehyde by dihydroxyacetone kinase-
catalyzed phosphorylation of D-glyceraldehyde to D-glyceraldehyde 3-
phosphate.
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31P NMR-spectra were recorded in hourly intervals as described above
for the first 14 hours and after 25 h. For quantification, the sum of the
integrals of the arising glyceraldehyde 3-phosphate (D/L-GAP), phospho-
enolpyruvic acid (PEP), and inorganic phosphate (Pi) were normalized to
100 and the particular values were plotted against the reaction time.
The discovery of dihydroxyacetone kinase as an enantio-
complementary enzyme to glycerol kinase, which catalyzes only
the phosphorylation of L-glyceraldehyde, is very valuable for a
highly selective phosphorylation of D-glyceraldehyde. It is also
interesting that the dihydroxyacetone kinase from C. freundii and
the glycerol kinase from E. coli are both composed of subunits of
similar size and contain the amino acid motif G-K-G as the
central part of the putative ATP-binding site. Dihydroxyacetone
kinase exists however as a dimer, while glycerol kinase forms a
tetramer [15].
Acknowledgements
We thank Roland Meier and Dr. Bernhard Schoenenberger for
TLC-analysis of enzymatic phosphorylation reactions. We also
would like to thank the German Federal Ministry of Education
and Research (BMBF) for the support of project P28 under the
cluster of Biocatalysis2021. Work at CSIC has been supported
by the grant MAT2015-65184-C2-2-R (MINECO/FEDER). COST
Action: CM1303 “Systems Biocatalysis” is acknowledged.
The recent discovery of
a glyceraldehyde-phosphorylating
function to a human triokinase [16] shows the importance of this
enzyme function in D-fructose utilization for human health and is
of much interest to the molecular understanding of the Hers
metabolic pathway [17].
Keywords: D-Glyceraldehyde 3-phosphate • Dihydroxyacetone
kinase • 31P-NMR • Biocatalysis • Enzymatic Phosphorylation
Experimental Section
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using an AKTA-FPLC system (GE Healthcare Life Science). In each
standard purification, 5 ml of cell free extract from induced bacteria
cultures were loaded on a HiLoad 26/60 Superdex 200 PG column.
Purification was carried out in 50 mM phosphate buffer pH 7.2 containing
NaCl (0.15 M) at a constant flow rate of 1.0 ml/min. Only central fractions
in the DHAK peak were taken in order to get the high-purity enzyme
required for D-GAP synthesis. Fractions containing DHAK were pooled
together in a dialysis membrane and incubated overnight at 4◦C in 3 L of
Tris-HCl buffer 5 mM (pH 7.2), under stirring. The dialyzed protein was
freeze-dried in glass vials (15 ml) and stored at 4◦C.
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Biocatalytic phosphorylations cataly-
zed by recombinant dihydroxyaceto-
ne kinase have been investigated with
quantitative 31P-NMR and pyruvate-
Dominik Gauss, Israel Sanchez-Moreno,
Isabel Oroz-Guinea, Eduardo Garcia-
Junceda and Roland Wohlgemuth*
Page No. – Page No.
kinase-catalyzed
ATP-regeneration
starting with dihydroxyacetone as sub-
strate. A nearly 100% conversion of
D-glyceraldehyde to D-glyceraldehy-
de-3-phosphate has been found.
Practically no formation of glyceral-
dehyde 3-phosphate was observed
with L-glyceraldehyde as substrate.
Dihydroxyacetone kinase-catalyzed
Phosphorylation
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